Congenital disorders of glycosylation (CDG) encompass over 170 identified genetic mutations characterized by defects in the generation, modification, attachment of sugar building blocks to glycoproteins. Consequently, the cellular structures, immune function, and mucin secretion can be detrimentally affected. Symptoms can manifest in infancy, negatively impact multiple organ systems. One specific subtype of CDG involves the inactivation of SLC35A2 gene, which encodes a Golgi apparatus-located transporter. The SLC35A2 is responsible for shutting UDP-galactose into Golgi, providing galactose for conversion from glucosylceramide to lactosylceramide. Despite existing studies have explored the influences of SLC35A2-CDG on various body systems, the relationship between SLC35A2 inactivation and GI tract disorder remains unclear. Specifically, the role of SLC35A2 in glycosylation of GI tract is unknown.
My objective is to determine the functions of SLC35A2 in glycosylation of GI tract and how SLC35A2 inactivation leads to GI tract disorders. The SLC35A2 is highly expressed in epithelial cells, which form a protective barrier on the GI tract surface. This epithelial cell layer prevents the invasion of microorganisms and loss of protein. When the layer is destroyed, inflammation happens, leading to the eventual GI disorders. In the former studies, Kot et al. discovered the inactivation of SLC35A2 could promote the epithelial cells transit to mesenchymal, which have no junctions with each other.[5] My hypothesis is SLC35A2 is involved in the glycosylation of epithelial cells within GI tract, contributing to its health and integrity. Given the genetic and physiological similarities between humans and zebrafish, coupled with the transparency features, zebrafish is an excellent model for GI tract dysfunction study. The immunofluorescence assays will be applied to visualize and quantify the severity of GI tract disorders. The long-term goal is to offer fresh perspectives and novel treatments for glycosylation related GI tract disorders.
Aim1: Determining which amino acid on SLC35A2 plays an important for glycosylation in the GI tract.
Rationale: Discerning each amino acid's contribution to the function of SLC35A2 can provide the framework for subsequent investigations into the roles of SLC35A2 into GI tract disorders.
Approach: Sequence alignment using Mega ClustalW will identify conserved amino acids across species. CRISPR/Cas9 will be employed to induce mutations at S213F and G282R in zebrafish, aiming to verify human cell mutations can inactivate SLC35A2 in zebrafish as well.[6] Both wild type and mutant zebrafish will be screened for phenotypic changes using fluorescence intensity measurements. Subsequently, UDP-galactose concentrations in epithelial cells from zebrafish GI tracts will be quantified by high-performance liquid chromatography.
Hypothesis: Mutations at S213F and G282R inactivate SLC35A2 on zebrafish, leading to the glycosylation disorders and subsequent GI tract dysfunctions. The concentration of UDP-gal on both mutant zebrafish are higher than that of wild type zebrafish.
Aim2: Identifying genes related with SLC35A2 glycosylation pathway and homeostasis of GI tract.
Rationale: Disorders in GI tract can be traced to gene interactions modulated by SLC35A2. The changes in gene expression can provided deeper insight into its roles in maintaining epithelial structure and function within GI tract.
Approach: RNA sequencing of epithelial cells from both wild type and mutant zebrafish will be conducted to analyze gene expression levels. Heatmaps and volcano plots are utilized for data visualization, with further Gene Ontology and KEGG pathway analysis for up-regulation genes. Then targeted upregulation genes will be knocked out by CRISP/Cas 9 in both zebrafish models to assess changes in phenotype through fluorescence intensity measurements.
Hypothesis: Genes related to epithelial cells and tight junctions are downregulated, while mesenchymal markers are upregulated in mutant zebrafish. Knocking out these upregulated genes in mutants will not induce GI tract disorders.
Aim3: Quantifying glycoproteome variations between wild-type and G282R mutant zebrafish.
Rationale: Comparing glycoprotein concentration changes across the experimental groups, providing insights into the potential therapeutic pathways or glycoproteins targets to restore normal glycosylation and maintain intestinal integrity.
D-galactose is the only reported treatment for SLC35A2 – CDG. [7]
Approach: This experiment involves two experimental groups, and both conclude wild and mutant zebrafish models. The control group fed with a standard diet while the other with a D-galactose enriched diet. Both groups will be monitored for GI tract alterations. Epithelial cells will be isolated from the GI tract via enzymatic dissociation activity followed with trypsin and glycoproteases digestion. Then glycopeptides enrichment and purification assay are applied to obtain the pure glycopeptides mixture. Then glycopeptides samples from four zebrafish groups are separately labeled using the isobaric tags for iTRAQ method. After labeling, they are mixed to allow for precise quantification during Tandem mass spectrometry analysis.
Hypothesis: Compared with wild type, mutant zebrafish has lower lactosylceramide and higher glucosylceramide concentration. Moreover, D-galactose treatment modulates the glycoproteomic profile in mutant zebrafish, particularly increases the lactosylceramide peak intensities identified via mass spectrometry.
In conclusion, the mutations on S213F and G282R can induce the glycosylation disorders by epithelial to mesenchymal transition. But D-galactose intake and rescue the disorders. In the future, based on the up-regulation genes and glycoproteins found before, reverse chemical screening will be applied to find more effective treatment for SLC35A2 – CDG.
My objective is to determine the functions of SLC35A2 in glycosylation of GI tract and how SLC35A2 inactivation leads to GI tract disorders. The SLC35A2 is highly expressed in epithelial cells, which form a protective barrier on the GI tract surface. This epithelial cell layer prevents the invasion of microorganisms and loss of protein. When the layer is destroyed, inflammation happens, leading to the eventual GI disorders. In the former studies, Kot et al. discovered the inactivation of SLC35A2 could promote the epithelial cells transit to mesenchymal, which have no junctions with each other.[5] My hypothesis is SLC35A2 is involved in the glycosylation of epithelial cells within GI tract, contributing to its health and integrity. Given the genetic and physiological similarities between humans and zebrafish, coupled with the transparency features, zebrafish is an excellent model for GI tract dysfunction study. The immunofluorescence assays will be applied to visualize and quantify the severity of GI tract disorders. The long-term goal is to offer fresh perspectives and novel treatments for glycosylation related GI tract disorders.
Aim1: Determining which amino acid on SLC35A2 plays an important for glycosylation in the GI tract.
Rationale: Discerning each amino acid's contribution to the function of SLC35A2 can provide the framework for subsequent investigations into the roles of SLC35A2 into GI tract disorders.
Approach: Sequence alignment using Mega ClustalW will identify conserved amino acids across species. CRISPR/Cas9 will be employed to induce mutations at S213F and G282R in zebrafish, aiming to verify human cell mutations can inactivate SLC35A2 in zebrafish as well.[6] Both wild type and mutant zebrafish will be screened for phenotypic changes using fluorescence intensity measurements. Subsequently, UDP-galactose concentrations in epithelial cells from zebrafish GI tracts will be quantified by high-performance liquid chromatography.
Hypothesis: Mutations at S213F and G282R inactivate SLC35A2 on zebrafish, leading to the glycosylation disorders and subsequent GI tract dysfunctions. The concentration of UDP-gal on both mutant zebrafish are higher than that of wild type zebrafish.
Aim2: Identifying genes related with SLC35A2 glycosylation pathway and homeostasis of GI tract.
Rationale: Disorders in GI tract can be traced to gene interactions modulated by SLC35A2. The changes in gene expression can provided deeper insight into its roles in maintaining epithelial structure and function within GI tract.
Approach: RNA sequencing of epithelial cells from both wild type and mutant zebrafish will be conducted to analyze gene expression levels. Heatmaps and volcano plots are utilized for data visualization, with further Gene Ontology and KEGG pathway analysis for up-regulation genes. Then targeted upregulation genes will be knocked out by CRISP/Cas 9 in both zebrafish models to assess changes in phenotype through fluorescence intensity measurements.
Hypothesis: Genes related to epithelial cells and tight junctions are downregulated, while mesenchymal markers are upregulated in mutant zebrafish. Knocking out these upregulated genes in mutants will not induce GI tract disorders.
Aim3: Quantifying glycoproteome variations between wild-type and G282R mutant zebrafish.
Rationale: Comparing glycoprotein concentration changes across the experimental groups, providing insights into the potential therapeutic pathways or glycoproteins targets to restore normal glycosylation and maintain intestinal integrity.
D-galactose is the only reported treatment for SLC35A2 – CDG. [7]
Approach: This experiment involves two experimental groups, and both conclude wild and mutant zebrafish models. The control group fed with a standard diet while the other with a D-galactose enriched diet. Both groups will be monitored for GI tract alterations. Epithelial cells will be isolated from the GI tract via enzymatic dissociation activity followed with trypsin and glycoproteases digestion. Then glycopeptides enrichment and purification assay are applied to obtain the pure glycopeptides mixture. Then glycopeptides samples from four zebrafish groups are separately labeled using the isobaric tags for iTRAQ method. After labeling, they are mixed to allow for precise quantification during Tandem mass spectrometry analysis.
Hypothesis: Compared with wild type, mutant zebrafish has lower lactosylceramide and higher glucosylceramide concentration. Moreover, D-galactose treatment modulates the glycoproteomic profile in mutant zebrafish, particularly increases the lactosylceramide peak intensities identified via mass spectrometry.
In conclusion, the mutations on S213F and G282R can induce the glycosylation disorders by epithelial to mesenchymal transition. But D-galactose intake and rescue the disorders. In the future, based on the up-regulation genes and glycoproteins found before, reverse chemical screening will be applied to find more effective treatment for SLC35A2 – CDG.
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Reference:
[1]Philadelphia, C. H. O. (n.d.). Congenital Disorders of glycosylation (CDG). Children’s Hospital of Philadelphia. https://www.chop.edu/conditions-diseases/congenital-disorders-glycosylation-cdg
[2]Home | CDG Hub. (n.d.). https://www.cdghub.com/
[3]Verheijen, J., Tahata, S., Kozicz, T., Witters, P., & Morava, É. (2020). Therapeutic approaches in Congenital Disorders of Glycosylation (CDG) involving N-linked glycosylation: an update. Genetics in Medicine, 22(2), 268–279. https://doi.org/10.1038/s41436-019-0647-2
[4] Dörre, K., Olczak, M., Wada, Y., Sosicka, P., Grüneberg, M., Reunert, J., Kurlemann, G., Fiedler, B., Biskup, S., Hörtnagel, K., Rust, S., & Marquardt, T. (2015). A new case of UDP‐galactose transporter deficiency (SLC35A2‐CDG): molecular basis, clinical phenotype, and therapeutic approach. Journal of Inherited Metabolic Disease, 38(5), 931–940. https://doi.org/10.1007/s10545-015-9828-6
[5]Kot, M., Mazurkiewicz, E., Wiktor, M., Wiertelak, W., Mazur, A. J., Rahalevich, A., Olczak, M., & Maszczak‐Seneczko, D. (2022). SLC35A2 deficiency promotes an Epithelial-to-Mesenchymal transition-like phenotype in Madin–Darby canine kidney cells. Cells, 11(15), 2273. https://doi.org/10.3390/cells11152273
[6]Li, D., & Mukhopadhyay, S. (2019). Functional analyses of the UDP-galactose transporter SLC35A2 using the binding of bacterial Shiga toxins as a novel activity assay. Glycobiology, 29(6), 490–503. https://doi.org/10.1093/glycob/cwz016
[7] SLC35A2-congenital disorder of glycosylation (SLC35A2-CDG) (no date) Frontiers in Congenital Disorders of Glycosylation. Available at: https://fcdgc.rarediseasesnetwork.org/diseases-studied/slc35a2-cdg (Accessed: 15 April 2024).
Image Reference:
https://www.verywellhealth.com/digestive-system-diseases-7375274
[1]Philadelphia, C. H. O. (n.d.). Congenital Disorders of glycosylation (CDG). Children’s Hospital of Philadelphia. https://www.chop.edu/conditions-diseases/congenital-disorders-glycosylation-cdg
[2]Home | CDG Hub. (n.d.). https://www.cdghub.com/
[3]Verheijen, J., Tahata, S., Kozicz, T., Witters, P., & Morava, É. (2020). Therapeutic approaches in Congenital Disorders of Glycosylation (CDG) involving N-linked glycosylation: an update. Genetics in Medicine, 22(2), 268–279. https://doi.org/10.1038/s41436-019-0647-2
[4] Dörre, K., Olczak, M., Wada, Y., Sosicka, P., Grüneberg, M., Reunert, J., Kurlemann, G., Fiedler, B., Biskup, S., Hörtnagel, K., Rust, S., & Marquardt, T. (2015). A new case of UDP‐galactose transporter deficiency (SLC35A2‐CDG): molecular basis, clinical phenotype, and therapeutic approach. Journal of Inherited Metabolic Disease, 38(5), 931–940. https://doi.org/10.1007/s10545-015-9828-6
[5]Kot, M., Mazurkiewicz, E., Wiktor, M., Wiertelak, W., Mazur, A. J., Rahalevich, A., Olczak, M., & Maszczak‐Seneczko, D. (2022). SLC35A2 deficiency promotes an Epithelial-to-Mesenchymal transition-like phenotype in Madin–Darby canine kidney cells. Cells, 11(15), 2273. https://doi.org/10.3390/cells11152273
[6]Li, D., & Mukhopadhyay, S. (2019). Functional analyses of the UDP-galactose transporter SLC35A2 using the binding of bacterial Shiga toxins as a novel activity assay. Glycobiology, 29(6), 490–503. https://doi.org/10.1093/glycob/cwz016
[7] SLC35A2-congenital disorder of glycosylation (SLC35A2-CDG) (no date) Frontiers in Congenital Disorders of Glycosylation. Available at: https://fcdgc.rarediseasesnetwork.org/diseases-studied/slc35a2-cdg (Accessed: 15 April 2024).
Image Reference:
https://www.verywellhealth.com/digestive-system-diseases-7375274