Orhan Kerim Inci, Andrés Felipe Leal, Nurselin Ates, Diego A Súarez, Angela Johana Espejo-Mojica, Carlos Javier Alméciga-Diaz, Volkan Seyrantepe
{"title":"酵母产生的重组人溶酶体β-己糖氨酸酶有效地挽救了Tay-Sachs病中GM2神经节苷脂的积累。","authors":"Orhan Kerim Inci, Andrés Felipe Leal, Nurselin Ates, Diego A Súarez, Angela Johana Espejo-Mojica, Carlos Javier Alméciga-Diaz, Volkan Seyrantepe","doi":"10.3390/jpm15050196","DOIUrl":null,"url":null,"abstract":"<p><p><b>Background:</b> Tay-Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the <i>HEXA</i> gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast <i>Pichia pastoris</i>, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. <b>Methods:</b> Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, <i>GM2AP</i> and <i>HEXB</i> expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. <b>Results:</b> Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the <i>HEXB</i> gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. <b>Conclusions:</b> Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells.</p>","PeriodicalId":16722,"journal":{"name":"Journal of Personalized Medicine","volume":"15 5","pages":""},"PeriodicalIF":3.0000,"publicationDate":"2025-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12113087/pdf/","citationCount":"0","resultStr":"{\"title\":\"Yeast-Produced Human Recombinant Lysosomal β-Hexosaminidase Efficiently Rescues GM2 Ganglioside Accumulation in Tay-Sachs Disease.\",\"authors\":\"Orhan Kerim Inci, Andrés Felipe Leal, Nurselin Ates, Diego A Súarez, Angela Johana Espejo-Mojica, Carlos Javier Alméciga-Diaz, Volkan Seyrantepe\",\"doi\":\"10.3390/jpm15050196\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><b>Background:</b> Tay-Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the <i>HEXA</i> gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast <i>Pichia pastoris</i>, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. <b>Methods:</b> Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, <i>GM2AP</i> and <i>HEXB</i> expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. <b>Results:</b> Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the <i>HEXB</i> gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. <b>Conclusions:</b> Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells.</p>\",\"PeriodicalId\":16722,\"journal\":{\"name\":\"Journal of Personalized Medicine\",\"volume\":\"15 5\",\"pages\":\"\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2025-05-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12113087/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Personalized Medicine\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3390/jpm15050196\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"HEALTH CARE SCIENCES & SERVICES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Personalized Medicine","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3390/jpm15050196","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"HEALTH CARE SCIENCES & SERVICES","Score":null,"Total":0}
Yeast-Produced Human Recombinant Lysosomal β-Hexosaminidase Efficiently Rescues GM2 Ganglioside Accumulation in Tay-Sachs Disease.
Background: Tay-Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells.
期刊介绍:
Journal of Personalized Medicine (JPM; ISSN 2075-4426) is an international, open access journal aimed at bringing all aspects of personalized medicine to one platform. JPM publishes cutting edge, innovative preclinical and translational scientific research and technologies related to personalized medicine (e.g., pharmacogenomics/proteomics, systems biology). JPM recognizes that personalized medicine—the assessment of genetic, environmental and host factors that cause variability of individuals—is a challenging, transdisciplinary topic that requires discussions from a range of experts. For a comprehensive perspective of personalized medicine, JPM aims to integrate expertise from the molecular and translational sciences, therapeutics and diagnostics, as well as discussions of regulatory, social, ethical and policy aspects. We provide a forum to bring together academic and clinical researchers, biotechnology, diagnostic and pharmaceutical companies, health professionals, regulatory and ethical experts, and government and regulatory authorities.