{"title":"小鼠细胞毒性T细胞系CTLL-2中溶酶体相关膜蛋白1和穿孔蛋白杂交分子的表达和定位","authors":"Yuto Ueno, Rikako Yamaguchi, Yumiko Tanaka, Yasunobu Miyake, Keiko Takaki, Ayaka Nakao, Ryota Ozaki, Yuka Yokota, Itsuki Fukai, Sara Kuwada, Natsuki Fukuoka, Naoaki Okuda, Ai Nishida, Tomoo Shiba, Takao Kataoka","doi":"10.1007/s10616-025-00794-8","DOIUrl":null,"url":null,"abstract":"<p><p>Cytotoxic T lymphocytes (CTL) and natural killer cells harbor lytic granules as secretory lysosomes. Lytic granules contain perforin as a soluble protein that forms pores and is essential for target cell lysis. Lysosomal-associated membrane protein 1 (LAMP1) is a single transmembrane protein that is enriched in lysosomes and is also present in lytic granules. In this study, to investigate the feasibility of membrane-integral markers for lytic granules, LAMP1- and perforin-based hybrid molecules were constructed and their expression and localization were examined in the murine cytotoxic T-cell line CTLL-2. Stable CTLL-2 transfectants were established by nucleofection using linearized vectors encoding human LAMP1 (1-417) fused to enhanced green fluorescent protein (EGFP) and FLAG (LAMP1-EGFP-FLAG) and human perforin (1-555) fused to human LAMP1 (325-417), EGFP, and FLAG (Perforin-LAMP1-EGFP-FLAG). Confocal microscopy showed that LysoTracker Red colocalized with LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG, and Perforin-LAMP1-EGFP-FLAG showed a broader cytoplasmic distribution than LAMP1-EGFP-FLAG. A Percoll density gradient centrifugation analysis revealed that LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG were distributed in fractions associated with the endogenous expression of perforin and LAMP1. The effects of glycosylation inhibitors on the expression and intracellular transport of LAMP1-EGFP-FLAG were also investigated. Concanamycin A, an inhibitor of vacuolar-type H<sup>+</sup>-ATPase, was unable to induce the proteolytic degradation of Perforin-LAMP1-EGFP-FLAG, in contrast to perforin. The present results demonstrated that some LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG localized to acidic granules or dense granules containing perforin and LAMP1 in CTLL-2 cells.</p>","PeriodicalId":10890,"journal":{"name":"Cytotechnology","volume":"77 4","pages":"129"},"PeriodicalIF":2.0000,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12198104/pdf/","citationCount":"0","resultStr":"{\"title\":\"Expression and localization of lysosomal-associated membrane protein 1- and perforin-based hybrid molecules in the murine cytotoxic T cell line CTLL-2.\",\"authors\":\"Yuto Ueno, Rikako Yamaguchi, Yumiko Tanaka, Yasunobu Miyake, Keiko Takaki, Ayaka Nakao, Ryota Ozaki, Yuka Yokota, Itsuki Fukai, Sara Kuwada, Natsuki Fukuoka, Naoaki Okuda, Ai Nishida, Tomoo Shiba, Takao Kataoka\",\"doi\":\"10.1007/s10616-025-00794-8\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Cytotoxic T lymphocytes (CTL) and natural killer cells harbor lytic granules as secretory lysosomes. Lytic granules contain perforin as a soluble protein that forms pores and is essential for target cell lysis. Lysosomal-associated membrane protein 1 (LAMP1) is a single transmembrane protein that is enriched in lysosomes and is also present in lytic granules. In this study, to investigate the feasibility of membrane-integral markers for lytic granules, LAMP1- and perforin-based hybrid molecules were constructed and their expression and localization were examined in the murine cytotoxic T-cell line CTLL-2. Stable CTLL-2 transfectants were established by nucleofection using linearized vectors encoding human LAMP1 (1-417) fused to enhanced green fluorescent protein (EGFP) and FLAG (LAMP1-EGFP-FLAG) and human perforin (1-555) fused to human LAMP1 (325-417), EGFP, and FLAG (Perforin-LAMP1-EGFP-FLAG). Confocal microscopy showed that LysoTracker Red colocalized with LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG, and Perforin-LAMP1-EGFP-FLAG showed a broader cytoplasmic distribution than LAMP1-EGFP-FLAG. A Percoll density gradient centrifugation analysis revealed that LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG were distributed in fractions associated with the endogenous expression of perforin and LAMP1. The effects of glycosylation inhibitors on the expression and intracellular transport of LAMP1-EGFP-FLAG were also investigated. Concanamycin A, an inhibitor of vacuolar-type H<sup>+</sup>-ATPase, was unable to induce the proteolytic degradation of Perforin-LAMP1-EGFP-FLAG, in contrast to perforin. The present results demonstrated that some LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG localized to acidic granules or dense granules containing perforin and LAMP1 in CTLL-2 cells.</p>\",\"PeriodicalId\":10890,\"journal\":{\"name\":\"Cytotechnology\",\"volume\":\"77 4\",\"pages\":\"129\"},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2025-08-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12198104/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cytotechnology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s10616-025-00794-8\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/6/25 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cytotechnology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s10616-025-00794-8","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/6/25 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Expression and localization of lysosomal-associated membrane protein 1- and perforin-based hybrid molecules in the murine cytotoxic T cell line CTLL-2.
Cytotoxic T lymphocytes (CTL) and natural killer cells harbor lytic granules as secretory lysosomes. Lytic granules contain perforin as a soluble protein that forms pores and is essential for target cell lysis. Lysosomal-associated membrane protein 1 (LAMP1) is a single transmembrane protein that is enriched in lysosomes and is also present in lytic granules. In this study, to investigate the feasibility of membrane-integral markers for lytic granules, LAMP1- and perforin-based hybrid molecules were constructed and their expression and localization were examined in the murine cytotoxic T-cell line CTLL-2. Stable CTLL-2 transfectants were established by nucleofection using linearized vectors encoding human LAMP1 (1-417) fused to enhanced green fluorescent protein (EGFP) and FLAG (LAMP1-EGFP-FLAG) and human perforin (1-555) fused to human LAMP1 (325-417), EGFP, and FLAG (Perforin-LAMP1-EGFP-FLAG). Confocal microscopy showed that LysoTracker Red colocalized with LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG, and Perforin-LAMP1-EGFP-FLAG showed a broader cytoplasmic distribution than LAMP1-EGFP-FLAG. A Percoll density gradient centrifugation analysis revealed that LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG were distributed in fractions associated with the endogenous expression of perforin and LAMP1. The effects of glycosylation inhibitors on the expression and intracellular transport of LAMP1-EGFP-FLAG were also investigated. Concanamycin A, an inhibitor of vacuolar-type H+-ATPase, was unable to induce the proteolytic degradation of Perforin-LAMP1-EGFP-FLAG, in contrast to perforin. The present results demonstrated that some LAMP1-EGFP-FLAG and Perforin-LAMP1-EGFP-FLAG localized to acidic granules or dense granules containing perforin and LAMP1 in CTLL-2 cells.
期刊介绍：
The scope of the Journal includes:
1. The derivation, genetic modification and characterization of cell lines, genetic and phenotypic regulation, control of cellular metabolism, cell physiology and biochemistry related to cell function, performance and expression of cell products.
2. Cell culture techniques, substrates, environmental requirements and optimization, cloning, hybridization and molecular biology, including genomic and proteomic tools.
3. Cell culture systems, processes, reactors, scale-up, and industrial production. Descriptions of the design or construction of equipment, media or quality control procedures, that are ancillary to cellular research.
4. The application of animal/human cells in research in the field of stem cell research including maintenance of stemness, differentiation, genetics, and senescence, cancer research, research in immunology, as well as applications in tissue engineering and gene therapy.
5. The use of cell cultures as a substrate for bioassays, biomedical applications and in particular as a replacement for animal models.
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