Glycosylation Pathway Research Articles

GlycoMaple: recent updates and applications in visualization and analysis of glycosylation pathways.

Post-translational modifications including glycosylation, phosphorylation, and lipidation expand the functionality and diversity of proteins. Protein glycosylation is one of the most abundant post-translational modifications in mammalian cells. The glycosylation process is regulated at multiple steps, including transcription, translation, protein folding, intracellular transport, and localization, and activity of glycosyltransferases and glycoside hydrolases. The glycosylation process is also affected by the concentration of sugar nucleotides in the lumen of the Golgi apparatus. Unlike the synthesis of other biological macromolecules, such as nucleic acids and proteins, glycan biosynthesis is not a template-driven process. In addition, the chemical complexity of glycan structures makes the glycosylation network extraordinarily intricate. We previously developed a web-based tool specially focused on glycan metabolic pathways known as GlycoMaple, which is able to easily visualize and estimate glycosylation pathways based on gene expression data. We recently updated GlycoMaple to incorporate the new genes and glycosylation pathways. Here, we introduce and discuss the uses and upgrades of GlycoMaple.

Application of a risk score model based on glycosylation-related genes in the prognosis and treatment of patients with low-grade glioma.

Low-grade gliomas (LGG) represent a heterogeneous and complex group of brain tumors. Despite significant progress in understanding and managing these tumors, there are still many challenges that need to be addressed. Glycosylation, a common post-translational modification of proteins, plays a significant role in tumor transformation. Numerous studies have demonstrated a close relationship between glycosylation modifications and tumor progression. However, the biological function of glycosylation-related genes in LGG remains largely unexplored. Their potential roles within the LGG microenvironment are also not well understood. We collected RNA-seq data and scRNA-seq data from patients with LGG from TCGA and GEO databases. The glycosylation pathway activity scores of each cluster and each patient were calculated by irGSEA and GSVA algorithms, and the differential genes between the high and low glycosylation pathway activity score groups were identified. Prognostic risk profiles of glycosylation-related genes were constructed using univariate Cox and LASSO regression analyses and validated in the CGGA database. An 8 genes risk score signature including ASPM, CHI3L1, LILRA4, MSN, OCIAD2, PTGER4, SERPING1 and TNFRSF12A was constructed based on the analysis of glycosylation-related genes. Patients with LGG were divided into high risk and low risk groups according to the median risk score. Significant differences in immunological characteristics, TIDE scores, drug sensitivity, and immunotherapy response were observed between these groups. Additionally, survival analysis of clinical medication information in the TCGA cohort indicated that high risk and low risk groups have different sensitivities to drug therapy. The risk score characteristics can thus guide clinical medication decisions for LGG patients. Our study established glycosylation-related gene risk score signatures, providing new perspectives and approaches for prognostic prediction and treatment of LGG.

N-glycoproteomic and proteomic alterations in SRD5A3-deficient fibroblasts.

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P<0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder.

Hyperglycosylation as an Indicator of Aging in the Bone Metabolome of Oryzias latipes.

Chronological aging of bone tissues is a multi-faceted process that involves a complex interplay of cellular, biochemical, and molecular mechanisms. Metabolites play a crucial role for bone homeostasis, and a changed metabolome is indicative for bone aging, although bone metabolomics are currently understudied. The vertebral bone metabolome of the model fish Japanese medaka (Oryzias latipes) was employed to identify sex-specific markers of bone aging. 265 and 213 metabolites were differently expressed in 8-month-old vs. 3-month-old female and male fish, respectively. The untargeted metabolomics pathway enrichment analysis indicated a sex-independent increased hyperglycosylation in 8-month-old individuals. The upregulated glycosylation pathways included glycosphingolipids, glycosylphosphatidylinositol anchors, O-glycans, and N-glycans. UDP-sugars and sialic acid were found to be major drivers in regulating glycosylation pathways and metabolic flux. The data indicate a disruption of protein processing at the endoplasmic reticulum and changes in O-glycan biosynthesis. Dysregulation of glycosylation, particularly through the hexosamine biosynthetic pathway, may contribute to bone aging and age-related bone loss. The results warrant further investigation into the functional involvement of increased glycosylation in bone aging. The potential of glycan-based biomarkers as early warning systems for bone aging should be explored and would aid in an advanced understanding of the progression of bone diseases such as osteoporosis.

GlyCompute: towards the automated analysis of protein N-linked glycosylation kinetics via an open-source computational framework.

Understanding the complex biosynthetic pathways of glycosylation is crucial for the expanding field of glycosciences. Computer-aided glycosylation analysis has greatly benefited in recent years from the development of tools found in web-based portals and open-source libraries. However, the in silico analysis of cellular glycosylation kinetics is underrepresented in current glycoscience-related tools and databases. This could be partly attributed to the limited accessibility of kinetic models developed using proprietary software and the difficulty in reliably parameterising such models. This work aims to address these challenges by proposing GlyCompute, an open-source framework demonstrating a novel, streamlined approach for the assembly, simulation, and parameterisation of kinetic models of protein N-linked glycosylation. Specifically, given one or more sets of experimentally observed N-glycan structures and their relative abundances, minimum representations of a glycosylation reaction network are generated. The topology of the resulting networks is then used to automatically assemble the material balances and kinetic mechanisms underpinning the mathematical model. To match the experimentally observed relative abundances, a sequential parameter estimation strategy using Bayesian inference is proposed, with stages determined automatically based on the underlying network topology. The proposed framework was tested on a case study involving the simultaneous fitting of the kinetic model to two protein N-linked glycoprofiles produced by the same CHO cell culture, showing good agreement with experimental observations. We envision that GlyCompute could help glycoscientists gain quantitative insights into the effect of enzyme kinetics and their perturbations on experimentally observed glycoprofiles in biomanufacturing and clinical settings.

Evolutionary rate covariation is pervasive between glycosylation pathways and points to potential disease modifiers.

Mutations in glycosylation pathways, such as N-linked glycosylation, O-linked glycosylation, and GPI anchor synthesis, lead to Congenital Disorders of Glycosylation (CDG). CDG typically present with seizures, hypotonia, and developmental delay but display large clinical variability with symptoms affecting every system in the body. This variability suggests modifier genes might influence the phenotypes. Because of the similar physiology and clinical symptoms, there are likely common genetic modifiers between CDG. Here, we use evolution as a tool to identify common modifiers between CDG and glycosylation genes. Protein glycosylation is evolutionarily conserved from yeast to mammals. Evolutionary rate covariation (ERC) identifies proteins with similar evolutionary rates that indicate shared biological functions and pathways. Using ERC, we identified strong evolutionary rate signatures between proteins in the same and different glycosylation pathways. Genome-wide analysis of proteins showing significant ERC with GPI anchor synthesis proteins revealed strong signatures with ncRNA modification proteins and DNA repair proteins. We also identified strong patterns of ERC based on cellular sub-localization of the GPI anchor synthesis enzymes. Functional testing of the highest scoring candidates validated genetic interactions and identified novel genetic modifiers of CDG genes. ERC analysis of disease genes and biological pathways allows for rapid prioritization of potential genetic modifiers, which can provide a better understanding of disease pathophysiology and novel therapeutic targets.

(Key1-001) congenital disorders of glycosylation: Glycobiology at the bedside.

Congenital disorders of glycosylation (CDG) are a group of rare monogenic human disorders caused by defects in the genes encoding the proteins that generate, attach, and modify glycans, thus disrupting cellular glycosylation machinery. Over 200 CDG caused by disruptions of 189 different genes are currently known. The multi-system disease manifestations of the CDG disorders highlight the importance of glycosylation across the organ systems. Clinical manifestations of CDG tend to group among genes contributing to the same glycosylation pathways, suggesting shared pathophysiology related to the glycosylation disruptions. However, the underlying glycosylation disruptions and pathophysiologic mechanisms responsible for specific CDG clinical manifestations have been determined for only a few hypoglycosylated proteins. The Frontiers in CDG Consortium (FCDGC) is an international network of clinical sites, laboratories, and patient advocacy groups established in 2019 to improve clinical symptoms, quality of life, and life expectancy for individuals with CDG. FCDGC seeks to answer decades of unresolved questions, address knowledge gaps, develop and validate new biochemical diagnostic techniques and therapeutic biomarkers, and explore novel therapeutic options for CDG. Over the past 5years, FCDGC has launched a Natural History Study with over 300 CDG patients, discovered novel biomarkers suggesting new mechanisms of disease, and launched clinical trials aiming to restore appropriate glycosylation and targeting newly identified potential mechanisms of disease. Technical advances in glycobiology are making it increasingly possible to comprehensively catalog glycoproteomic data and to probe functional impact of altered glycosylation. My laboratory applies glycoproteomic technologies to samples from human subjects and genetic model systems to identify glycosylation abnormalities and unlock new insights from translational glycobiology. Current findings and accomplishments highlight the ongoing bottlenecks and knowledge gaps at intersections of glycobiology and clinical care requiring further investigation.

Dual Glycosyltransferases from Campylobacter concisus Diverge from the Canonical Campylobacter N-Linked Glycan Assembly Pathway.

Species within the Campylobacter genus are recognized as emerging human pathogens. Common to all known members of the genus is the presence of an asparagine-linked glycosylation pathway encoded by the pgl operon. Campylobacter species are divided into two major groups, Group I and Group II. To date, most biochemical studies have focused on the Group I species including Campylobacter jejuni. We recently reported that the Group II Campylobacter concisus pathway deviates from that of Group I by the inclusion of a C-6″-oxidized GalNAc (GalNAcA) at the third position installed by PglJ. Herein, we investigate the diversification of the PglH enzymes that act subsequent to installation of GalNAcA. The majority of pgl operons from Group II species, including C. concisus, encode two GT-B fold glycosyltransferases (GTs), PglH1 and PglH2. As the functions of these GTs were not clear by simple comparison of their sequences to that of C. jejuni PglH, further analyses were required. We show that subsequent to the action of PglJ, PglH2 installs the next HexNAc followed by PglH1 adding a single sugar. These steps diverge from the C. jejuni pathway not only in the identity of the sugar donors (UDP-GlcNAc) but also in installing single sugars rather than acting processively. These biochemical studies were extended via bioinformatics to identify sequence signatures that provide predictive capabilities for unraveling the prokaryotic glycan landscape. Phylogenetic analysis showed early divergence between the C. jejuni PglH orthologs and C. concisus PglH1/PglH2 orthologs, leading to diversification of the final glycan.

Metabolism of isodecyl diphenyl phosphate in rice and microbiome system: Differential metabolic pathways and underlying mechanisms

Isodecyl diphenyl phosphate (IDDP) is among the emerging aromatic organophosphate esters (aryl-OPEs) that pose risks to both human beings and other organisms. This study aims to investigate the translocation and biotransformation behavior of IDDP in rice and the rhizosphere microbiome through hydroponic exposure (the duration of hydroponic exposure was 10 days). The rhizosphere microbiome 9-FY was found to efficiently eliminate IDDP, thereby reducing its uptake in rice tissues and mitigating the negative impact of IDDP on rice growth. Furthermore, this study proposed the first-ever transformation pathways of IDDP, identifying hydrolysis, hydroxylation, methylation, methoxylation, carboxylation, and glucuronidation products. Notably, the methylation and glycosylation pathways were exclusively observed in rice, indicating that the transformation of IDDP in rice may be more complex than in microbiome 9-FY. Additionally, the presence of the product COOH-IDDP in rice suggested that there might be an exchange of degradation products between rice and rhizobacteria, implying their potential interaction. This finding highlights the significance of rhizobacteria's role which cannot be overlooked in the accumulation and transformation of organic pollutants in grain crops. The study revealed active members in 9-FY during IDDP degradation, and metagenomic analysis indicated that most of the active populations contained IDDP-degrading genes. Moreover, transcriptome sequencing showed that cytochrome P450, acid phosphatase, glucosyltransferase, and methyltransferases genes in rice were up-regulated, which was further confirmed by RT-qPCR. This provides insight into the intermediate products identified in rice, such as hydrolysis, hydroxylated, glycosylated, and methylated products. These results significantly contribute to our understanding of the translocation and transformation of organophosphate esters (OPEs) in plants and the rhizosphere microbiome, and reveal the fate of OPEs in rice and microbiome system to ensure the paddy yield and rice safety.

Identification of CNTN2 as a genetic modifier of PIGA-CDG through pedigree analysis of a family with incomplete penetrance and functional testing in Drosophila.

Loss of function mutations in the X-linked PIGA gene lead to PIGA-CDG, an ultra-rare congenital disorder of glycosylation (CDG), typically presenting with seizures, hypotonia, and neurodevelopmental delay. We identified two brothers (probands) with PIGA-CDG, presenting with epilepsy and mild developmental delay. Both probands carry PIGA S132C , an ultra-rare variant predicted to be damaging. Strikingly, the maternal grandfather and a great-uncle also carry PIGA S132C , but neither presents with symptoms associated with PIGA-CDG. We hypothesized genetic modifiers may contribute to this reduced penetrance. Using whole genome sequencing and pedigree analysis, we identified possible susceptibility variants found in the probands and not in carriers and possible protective variants found in the carriers and not in the probands. Candidate variants included heterozygous, damaging variants in three genes also involved directly in GPI-anchor biosynthesis and a few genes involved in other glycosylation pathways or encoding GPI-anchored proteins. We functionally tested the predicted modifiers using a Drosophila eye-based model of PIGA-CDG. We found that loss of CNTN2 , a predicted protective modifier, rescues loss of PIGA in Drosophila eye-based model, like what we predict in the family. Further testing found that loss of CNTN2 also rescues patient-relevant phenotypes, including seizures and climbing defects in Drosophila neurological models of PIGA-CDG. By using pedigree information, genome sequencing, and in vivo testing, we identified CNTN2 as a strong candidate modifier that could explain the incomplete penetrance in this family. Identifying and studying rare disease modifier genes in human pedigrees may lead to pathways and targets that may be developed into therapies.

Manipulating mannose metabolism as a potential anticancer strategy.

Cancer cells acquire metabolic advantages over their normal counterparts regarding the use of nutrients for sustained cell proliferation and cell survival in the tumor microenvironment. Notable among the metabolic traits in cancer cells is the Warburg effect, which is a reprogrammed form of glycolysis that favors the rapid generation of ATP from glucose and the production of biological macromolecules by diverting glucose into various metabolic intermediates. Meanwhile, mannose, which is the C-2 epimer of glucose, has the ability to dampen the Warburg effect, resulting in slow-cycling cancer cells that are highly susceptible to chemotherapy. This anticancer effect of mannose appears when its catabolism is compromised in cancer cells. Moreover, de novo synthesis of mannose within cancer cells has also been identified as a potential target for enhancing chemosensitivity through targeting glycosylation pathways. The underlying mechanisms by which alterations in mannose metabolism induce cancer cell vulnerability are just beginning to emerge. This review summarizes the current state of our knowledge of mannose metabolism and provides insights into its manipulation as a potential anticancer strategy.

Nanoplastics exposure simplifies the network structure of sea cucumber (Apostichopus japonicus) gut microbiota and improves cluster randomness

Nanoplastics (NPs) are abundant in ocean environments, leading to environmental pollution and notable disruptions to the physiological functions of marine animals. To investigate the toxic effects of NPs on echinoderms, specifically sea cucumbers (Apostichopus japonicus), they were exposed to varying concentrations of NPs (0, 102, 104 particles/L) for 14 d. Subsequently, the 102 particles/L exposure group was purified for 35 d to elucidate the impact of both NPs exposure and purification on the intestinal bacteria structure and function. The results showed that the richness and variety of intestinal bacteria in sea cucumbers significantly reduced under NPs exposure, and then they could be restored to the pre-exposure treatment state after 35 d of purification. With the increase of NPs exposure concentration in the environment, the intestinal core bacteria gradually changed from Firmicutes and Proteobacteria to Pseudoalteromonas and Vibrio. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database annotated that the gut microbiota of sea cucumbers was significantly downregulated in the glycosylation, carbohydratic and amino acid metabolic pathways (P < 0. 05), exogenous substance biodegradation and metabolism, DNA replication and repair pathways were significantly up-regulated (P < 0.05) under the exposure of NPs. In addition, nanoplastics exposure simplified the symbiotic network relationships of the gut bacteria, reduced the selective effect of host on the intestinal bacteria, and increased stochasticity. In conclusion, waterborne NPs can adversely affect the structure and function of sea cucumber intestinal bacteria, with these effects persisting for a duration. However, as the purification time lengthens, these adverse effects gradually diminish. This study aims to provide some theoretical basis for the biotoxic effects of NPs.

Cell-based glycoengineering of extracellular vesicles through precise genome editing

Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a “simple cell” strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5/6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes.

Identification of two novel variants in ALG11 causing congenital disorder of glycosylation

BackgroundCongenital disorders of glycosylation (CDG) represent a heterogeneous group of rare inherited metabolic disorders due to abnormalities in protein or lipid glycosylation pathways, affecting multiple systems, and frequently being accompanied by neurological symptoms. ALG11-CDG, also known as CDG-1p, arises from a deficiency in a specific mannosyltransferase encoded by the ALG11 gene. To date, only 17 cases have been documented, and these patients have prominent clinical phenotypes, including seizures, developmental delay, and microcephaly. MethodsWe describe a novel case of a four-month-old boy from a Chinese family exhibiting developmental delay, seizures, and microcephaly. Trio whole-exome sequencing (WES) and subsequent Sanger sequencing were employed to identify the potential genetic cause, and functional study was performed to evaluate the pathogenicity of genetic variant identified. ResultsTrio WES unveiled novel compound heterozygous variants: c.1307G>T (p.G436V) and c.1403G>A (p.R468H) within exon 4 of the ALG11 gene, inherited from the father and mother, respectively. Subsequent in vitro functional analysis revealed decreased stability of the mutant protein and concurrent hypoglycosylation of GP130, a hyperglycosylated protein. ConclusionsOur findings not only expand the clinical and variant spectrum of ALG11-CDG, but also emphasize the importance of WES as a first-tier genetic test in determining the molecular diagnosis.

Dose–response relationship between serum N-glycan markers and liver fibrosis in chronic hepatitis B

BackgroundEvaluation of liver fibrosis played a monumental role in the diagnosis and monitoring of chronic hepatitis B (CHB). We aimed to explore the value of serum N-glycan markers in liver fibrosis.MethodsThis multi-center (33 hospitals) study recruited 760 treatment-naïve CHB patients who underwent liver biopsy. Serum N-glycan markers were analyzed by DNA sequencer-assisted fluorophore-assisted with capillary electrophoresis (DSA-FACE) technology. First, we explore the relationship between 12 serum N-glycan markers and the fibrosis stage. Then, we developed a Px score for diagnosing significant fibrosis using the LASSO regression. Next, we compared the diagnostic performances between Px, LSM, APRI, and FIB-4. Finally, we explored the relationships between glycosyltransferase gene and liver fibrosis with RNA-transcriptome sequencing.ResultsWe included 622 CHB participants: male-dominated (69.6%); median age 42.0 (IQR 34.0–50.0); 287 with normal ALT; 73.0% with significant fibrosis. P5(NA2), P8(NA3), and P10(NA4) were opposite to the degree of fibrosis, while other profiles (except for P0[NGA2]) increased with the degree of fibrosis. Seven profiles (P1[NGA2F], P2[NGA2FB], P3[NG1A2F], P4[NG1A2F], P7[NA2FB], P8[NA3], and P9[NA3Fb]) were selected into Px score. Px score was associated with an increased risk of significant fibrosis (for per Px score increase, the risk of significant fibrosis was increased by 3.54 times (OR = 4.54 [2.63–7.82]) in the fully-adjusted generalized linear model. p for trend was <0.001. The diagnostic performance of the Px score was superior to others. Glycosyltransferase genes were overexpressed in liver fibrosis, and glycosylation and glycosyltransferase-related pathways were significantly enriched.ConclusionsSerum N-glycan markers were positively correlated with liver fibrosis. Px score had good performance in distinguishing significant fibrosis.

Tumor suppressive role of the antimicrobial lectin REG3A targeting the O -GlcNAc glycosylation pathway.

Antimicrobial proteins of the regenerating family member 3 alpha (REG3A) family provide a first line of protection against infections and transformed cells. Their expression is inducible by inflammation, which makes their role in cancer biology less clear since an immune-inflammatory context may preexist or coexist with cancer, as occurs in HCC. The aim of this study is to clarify the role of REG3A in liver carcinogenesis and to determine whether its carbohydrate-binding functions are involved. This study provides evidence for a suppressive role of REG3A in HCC by reducing O -GlcNAcylation in 2 mouse models of HCC, in vitro cell studies, and clinical samples. REG3A expression in hepatocytes significantly reduced global O -GlcNAcylation and O -GlcNAcylation of c-MYC in preneoplastic and tumor livers and markedly inhibited HCC development in REG3A-c-MYC double transgenic mice and mice exposed to diethylnitrosamine. REG3A modified O -GlcNAcylation without altering the expression or activity of O-linked N-acetylglucosaminyltransferase, O-linked N-acetylglucosaminyl hydrolase, or glutamine fructose-6-phosphate amidotransferase. Reduced O -GlcNAcylation was consistent with decreased levels of UDP-GlcNAc in precancerous and cancerous livers. This effect was linked to the ability of REG3A to bind glucose and glucose-6 phosphate, suggested by a REG3A mutant unable to bind glucose and glucose-6 phosphate and alter O -GlcNAcylation. Importantly, patients with cirrhosis with high hepatic REG3A expression had lower levels of O -GlcNAcylation and longer cancer-free survival than REG3A-negative cirrhotic livers. REG3A helps fight liver cancer by reducing O -GlcNAcylation. This study suggests a new paradigm for the regulation of O -GlcNAc signaling in cancer-related pathways through interactions with the carbohydrate-binding function of REG3A.

MDB-61. STARVING TUMORS OF POLYAMINES: A NOVEL APPROACH IN TREATING AGGRESSIVE PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Polyamines, essential intracellular polycations, govern fundamental cell processes and drive oncogenesis. Our research unveiled the dependency of pediatric Diffuse Midline Gliomas (DMG) on the polyamine pathway (Khan et al.,2021). Disruption of this pathway with the synthesis inhibitor DFMO, and the transport inhibitor AMXT1501, potently inhibited DMG tumors, yielding unprecedented survival in-vivo. We hypothesized that dual polyamine pathway targeting could similarly suppress growth in other aggressive pediatric brain tumors. METHODS/RESULTS We analyzed the expression of polyamine pathway regulators in high-risk pediatric brain cancers ZERO precision medicine platform and found elevated expression in medulloblastoma. ODC1, a polyamine synthesis driver, was found to have significant correlation in high-grade gliomas with four key functional tumor states—stemness, cell cycle, hypoxia, and angiogenesis. Medulloblastoma cells were sensitive to treatment with DFMO but showed a compensatory increase in polyamine transporter expression. Conversely, treatment with AMXT1501 increased ODC1 expression. Dual targeting of the polyamine pathway potently and synergistically inhibited cell proliferation and clonogenic potential in Group-3 medulloblastoma cells. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT1501 in combination led to induction of apoptosis. Critically, dual polyamine inhibition potently sensitized medulloblastoma cells to the topoisomerase-I inhibitor, SN-38, leading to complete eradication of medulloblastoma cell proliferation and colony formation at nanomolar chemotherapeutic concentrations, with the effect most pronounced in Group-3 medulloblastoma. In triple combination-treated cells, over-representation analysis revealed heightened mitotic DNA damage, macroautophagy, and apoptosis, coupled with diminished neuron/synapse development, glycosylation, and cellular signaling pathways. We are currently testing this treatment strategy in aggressive orthotopic medulloblastoma models. CONCLUSION These findings suggest targeting the polyamine pathway as a promising strategy to sensitize aggressive pediatric medulloblastoma tumors to chemotherapy. DFMO/AMXT1501 is in Phase I evaluation for adults (NCT05500508) and advancing to a Phase I/II trial for children through the CONNECT consortium.

Early onset LGMDR19 with unusual features related to GMPPB gene in South Indian siblings with variable phenotype

Guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) is a cytoplasmic enzyme that catalyzes the synthesis of GDP-mannose, a crucial substrate for several glycosylation pathways. Reports of pathogenic variants in the GMPPB gene are infrequent. As of April 2023, 109 cases with pathogenic variants in the GMPPB gene have been reported worldwide. Here, we present two siblings born of consanguineous parentage from Southern India. This is a retrospective study on genetically confirmed siblings with GMPPB pathogenic variants. The siblings, a 15-year-old girl, and a 13-year-old boy, presented with progressive limb-girdle weakness and cataracts from early childhood. The girl had exertion-induced breathlessness and mental subnormality. Creatine kinase levels were 5750 and 4320 IU/L. An echocardiogram of the heart revealed global hypokinesia, moderate left ventricular dysfunction, and mild mitral regurgitation with dilation of the left atrium and left ventricle in the girl child. Exome sequencing showed a homozygous pathogenic variant (NM_021971.4 (GMPPB): c.358A&gt;G (p.Met120Val) in Exon 4 of GMPPB gene in both the siblings. Thus, we report an unusual early onset LGMD phenotype of GMPPB-related disorder with cardiac involvement and cataracts from a single family.

Genetics of glycosylation in mammalian development and disease.

Glycosylation of proteins and lipids in mammals is essential for embryogenesis and the development of all tissues. Analyses of glycosylation mutants in cultured mammalian cells and model organisms have been key to defining glycosylation pathways and the biological functions of glycans. More recently, applications of genome sequencing have revealed the breadth of rare congenital disorders of glycosylation in humans and the influence of genetics on the synthesis of glycans relevant to infectious diseases, cancer progression and diseases of the immune system. This improved understanding of glycan synthesis and functions is paving the way for advances in the diagnosis and treatment of glycosylation-related diseases, including the development of glycoprotein therapeutics through glycosylation engineering.

Liposome-encapsulated mannose-1-phosphate therapy improves global N-glycosylation in different congenital disorders of glycosylation

Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.