bims-meglyc 2024-05-19 papers

bims-meglyc

Biomed News

on Metabolic disorders affecting glycosylation

Issue of 2024–05–19
nine papers selected by
Silvia Radenkovic, UMC Utrecht

Liposome-encapsulated mannose-1-phosphate therapy improves global N-glycosylation in different congenital disorders of glycosylation.
D-mannose as a new therapy for fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG).
Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability.
Normal transferrin glycosylation does not rule out severe ALG1 deficiency.
Destabilization and Degradation of a Disease-Linked PGM1 Protein Variant.
Defining albumin as a glycoprotein with multiple N-linked glycosylation sites.
In silico simulation of glycosylation and related pathways.
Clinical Genetic and Genomic Testing in Congenital Heart Disease and Cardiomyopathy.
Generation of induced pluripotent stem cell lines from two unrelated patients affected by intellectual disability carrying homozygous variants in SGIP1.

Mol Genet Metab. 2024 May 07. pii: S1096-7192(24)00371-8. [Epub ahead of print]142(2): 108487

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

Rohit Budhraja, Silvia Radenkovic, Anu Jain, Irena J J Muffels, Moulay Hicham Alaoui Ismaili, Tamas Kozicz, Akhilesh Pandey, Eva Morava.

  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.

Keywords:  Congenital disorders of glycosylation; GLM101; Glycoproteomics; Glycosylation; PMM2-CDG; Therapy

DOI:  https://doi.org/10.1016/j.ymgme.2024.108487
Mol Genet Metab. 2024 May 09. pii: S1096-7192(24)00372-X. [Epub ahead of print]142(2): 108488

D-mannose as a new therapy for fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG).

Rodrigo Tzovenos Starosta, Angela J Lee, Elizabeth R Toolan, Miao He, Parith Wongkittichote, Earnest James Paul Daniel, Silvia Radenkovic, Rohit Budhraja, Akhilesh Pandey, Jaiprakash Sharma, Eva Morava, Hoanh Nguyen, Patricia I Dickson.

   INTRODUCTION: Fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG) is a rare autosomal recessive inborn error of metabolism characterized by a decreased flux through the salvage pathway of GDP-fucose biosynthesis due to a block in the recycling of L-fucose that exits the lysosome. FCSK-CDG has been described in 5 individuals to date in the medical literature, with a phenotype comprising global developmental delays/intellectual disability, hypotonia, abnormal myelination, posterior ocular disease, growth and feeding failure, immune deficiency, and chronic diarrhea, without clear therapeutic recommendations.
PATIENT AND METHODS: In a so far unreported FCSK-CDG patient, we studied proteomics and glycoproteomics in vitro in patient-derived fibroblasts and also performed in vivo glycomics, before and after treatment with either D-Mannose or L-Fucose.
RESULTS: We observed a marked increase in fucosylation after D-mannose supplementation in fibroblasts compared to treatment with L-Fucose. The patient was then treated with D-mannose at 850 mg/kg/d, with resolution of the chronic diarrhea, resolution of oral aversion, improved weight gain, and observed developmental gains. Serum N-glycan profiles showed an improvement in the abundance of fucosylated glycans after treatment. No treatment-attributed adverse effects were observed.
CONCLUSION: D-mannose is a promising new treatment for FCSK-CDG.

Keywords:  Aleuria aurantia; CDG; Chronic diarrhea; Congenital disorders of glycosylation; Developmental delay; Exome sequencing; Fucose; Fucose kinase; Glycoproteomics; Mannose

DOI:  https://doi.org/10.1016/j.ymgme.2024.108488
Mol Genet Metab. 2024 May 08. pii: S1096-7192(24)00376-7. [Epub ahead of print]142(2): 108492

Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability.

Marta Murray, Lindsay Davidson, Andrew T Ferenbach, Dirk Lefeber, Daan M F van Aalten.

  Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.

Keywords:  Development; Early development; O-GlcNAc; OGT-CDG; Patient derived IPSCs

DOI:  https://doi.org/10.1016/j.ymgme.2024.108492
JIMD Rep. 2024 May;65(3): 135-143

Normal transferrin glycosylation does not rule out severe ALG1 deficiency.

Inez Bosnyak, Mustafa Sadek, Wasantha Ranatunga, Tamas Kozicz, Eva Morava.

  ALG1-CDG is a rare, clinically variable metabolic disease, caused by the defect of adding the first mannose (Man) to N-acetylglucosamine (GlcNAc2)-pyrophosphate (PP)-dolichol to the growing oligosaccharide chain, resulting in impaired N-glycosylation of proteins. N-glycosylation has a key role in functionality, stability, and half-life of most proteins. Therefore, congenital defects of glycosylation typically are multisystem disorders. Here we report a 3-year-old patient with severe neurological, cardiovascular, respiratory, musculoskeletal and gastrointestinal symptoms. ALG1-CDG was suggested based on exome sequencing and Western blot analysis. Despite her severe clinical manifestations and genetic diagnosis, serum transferrin glycoform analysis was normal. Western blot analysis of highly glycosylated proteins in fibroblasts revealed decreased intercellular adhesion molecule 1 (ICAM1), but normal lysosomal associated membrane protein 1 and 2 (LAMP1 and LAMP2) expression levels. Glycoproteomics in fibroblasts showed the presence of the abnormal tetrasacharide. Reviewing the literature, we found 86 reported ALG1-CDG patients, but only one with normal transferrin analysis. Based on our results we would like to highlight the importance of multiple approaches in diagnosing ALG1-CDG, as normal serum transferrin glycosylation or other biomarkers with normal expression levels can occur.

Keywords:  ALG1; CDG; congenital disorders of glycosylation; transferrin

DOI:  https://doi.org/10.1002/jmd2.12415
Biochemistry. 2024 May 14.

Destabilization and Degradation of a Disease-Linked PGM1 Protein Variant.

Frederik Gouliaev, Nicolas Jonsson, Sarah Gersing, Michael Lisby, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen.

PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.

DOI: https://doi.org/10.1021/acs.biochem.4c00042
J Transl Med. 2024 May 13. 22(1): 454

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites.

Kishore Garapati, Anu Jain, Benjamin J Madden, Dong-Gi Mun, Jyoti Sharma, Rohit Budhraja, Akhilesh Pandey.

   BACKGROUND: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.
METHODS: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC-MS/MS.
RESULTS: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.
CONCLUSIONS: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Keywords:  BSA; Glycoproteomics; HSA; Human serum albumin; Microheterogeneity; Novel glycosylation site

DOI:  https://doi.org/10.1186/s12967-024-05000-5
Anal Bioanal Chem. 2024 May 15.

In silico simulation of glycosylation and related pathways.

Yukie Akune-Taylor, Akane Kon, Kiyoko F Aoki-Kinoshita.

  Glycans participate in a vast number of recognition systems in diverse organisms in health and in disease. However, glycans cannot be sequenced because there is no sequencer technology that can fully characterize them. There is no "template" for replicating glycans as there are for amino acids and nucleic acids. Instead, glycans are synthesized by a complicated orchestration of multitudes of glycosyltransferases and glycosidases. Thus glycans can vary greatly in structure, but they are not genetically reproducible and are usually isolated in minute amounts. To characterize (sequence) the glycome (defined as the glycans in a particular organism, tissue, cell, or protein), glycosylation pathway prediction using in silico methods based on glycogene expression data, and glycosylation simulations have been attempted. Since many of the mammalian glycogenes have been identified and cloned, it has become possible to predict the glycan biosynthesis pathway in these systems. By then incorporating systems biology and bioprocessing technologies to these pathway models, given the right enzymatic parameters including enzyme and substrate concentrations and kinetic reaction parameters, it is possible to predict the potentially synthesized glycans in the pathway. This review presents information on the data resources that are currently available to enable in silico simulations of glycosylation and related pathways. Then some of the software tools that have been developed in the past to simulate and analyze glycosylation pathways will be described, followed by a summary and vision for the future developments and research directions in this area.

Keywords:  Bioinformatics; Glycosylation; Pathways; Prediction; Software; Systems biology

DOI:  https://doi.org/10.1007/s00216-024-05331-8
J Clin Med. 2024 Apr 26. pii: 2544. [Epub ahead of print]13(9):

Clinical Genetic and Genomic Testing in Congenital Heart Disease and Cardiomyopathy.

Mahati Pidaparti, Gabrielle C Geddes, Matthew D Durbin.

  Congenital heart disease (CHD) and cardiomyopathies are the leading cause of morbidity and mortality worldwide. These conditions are often caused by genetic factors, and recent research has shown that genetic and genomic testing can provide valuable information for patient care. By identifying genetic causes, healthcare providers can screen for other related health conditions, offer early interventions, estimate prognosis, select appropriate treatments, and assess the risk for family members. Genetic and genomic testing is now the standard of care in patients with CHD and cardiomyopathy. However, rapid advances in technology and greater availability of testing options have led to changes in recommendations for the most appropriate testing method. Several recent studies have investigated the utility of genetic testing in this changing landscape. This review summarizes the literature surrounding the clinical utility of genetic evaluation in patients with CHD and cardiomyopathy.

Keywords:  cardiomyopathy 2; congenital heart disease 1; genetic testing 3; genome sequencing; genomic testing; personalized medicine

DOI:  https://doi.org/10.3390/jcm13092544
Stem Cell Res. 2024 May 09. pii: S1873-5061(24)00140-5. [Epub ahead of print]77 103442

Generation of induced pluripotent stem cell lines from two unrelated patients affected by intellectual disability carrying homozygous variants in SGIP1.

Lieke Dillen, Neelam Fatima, Marina P Hommersom, Ece Çepni, Fareeha Fatima, Ellen van Beusekom, Silvia Albert, Johanna M van Hagen, Bert B A de Vries, Asma Ali Khan, Arjan P M de Brouwer, Hans van Bokhoven.

Intellectual disability (ID) is a diverse neurodevelopmental condition and almost half of the cases have a genetic etiology. SGIP1 acts as an endocytic protein that influences the signaling of receptors in neuronal systems related to energy homeostasis through its interaction with endophilins. This study focuses on the generation and characterization of induced pluripotent stem cells (iPSC) from two unrelated patients due to a frameshift variant (c.764dupA, NM_032291.4) and a splice donor site variant (c.74 + 1G > A, NM_032291.4) in the SGIP1 gene.

DOI: https://doi.org/10.1016/j.scr.2024.103442