[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-586] Identification of ferritins associated with iron accumulation in chiton radular tissue

○Rui Onishi¹, Masahiro Shimizu², Koki Okada¹, Nicole Angeline Robles Vizconde¹, Tadayoshi Kanao³, Takashi Tamura³, Kenji Okoshi⁴, Kiori Obuse³, David Kisailus⁵, Akira Satoh³, Michiko Nemoto³ (1. Graduate School of Environmental, Life, Natural Science and Technology, Okayama University (Japan), 2. Laboratory of Radiation Material Science, Institute for Integrated Radiation and Nuclear Science, Kyoto University (Japan), 3. Faculty of Environmental, Life, Natural Science and Technology, Okayama University (Japan), 4. Department of Environmental Science, Faculty of Science, Toho University (Japan), 5. Department of Materials Science and Engineering, University of California, Irvine (USA))

Keywords:

iron,ferritin,mollusk,biomineralization

[Purpose]Chitons possess a feeding organ called the radula, whose teeth are composed of magnetite (Fe₃O₄). To form these magnetite teeth, chitons concentrate over 100 mg of iron per gram of radular tissue (Okoshi et al., 1996). The mechanism underlying this remarkable tissue-specific iron accumulation remains unclear. Elucidating how chitons concentrate iron at levels that are excessive for many other organisms may provide insight into the development of novel therapies for diseases associated with iron overload. Previous studies have shown that ferritin, an iron storage protein, is abundant in radular tissue and has been suggested to be involved in the transport of a large amount of iron (Shaw et al., 2009). However, the ferritin molecules responsible for this process have not been identified. Mollusks possess both cytoplasmic ferritin and secretory ferritin, the latter of which is released into the hemolymph. Recent transcriptome analyses have suggested that cytoplasmic ferritin genes are highly expressed in chiton radular tissue (Nemoto et al., 2019). In this study, we aimed to identify the major ferritin proteins present in radular tissue.[Method]The chiton Acanthopleura japonica was dissected, and radular tissue was isolated. Epithelial cells were collected from the radular tissue and disrupted by sonication. After heat treatment, the supernatant was collected by centrifugation and fractionated by size exclusion chromatography. The resulting fractions were analyzed by BN-PAGE and Tricine SDS-PAGE. Proteins were identified by LC-MS/MS following in-gel digestion. Transcriptome analysis was then performed on the tissues showing high expression of the identified ferritin genes. Ferritins were also purified from hemolymph using the same procedure used for radular tissue, and their subunits were identified. Ferritin sequences identified from each tissue were compared to identify motifs specific to radular tissue ferritins. Ferritin purified from chiton hemolymph was fluorescently labeled and injected into live chitons. Frozen sections of radular tissue were prepared and examined by fluorescence microscopy.[Results and Conclusion]LC-MS/MS analysis identified four ferritins in the radular tissue. Nine ferritins were identified in the hemolymph, including all four ferritins detected in the radular tissue. The corresponding ferritin genes were highly expressed in specific tissues. A motif specific to the radular-tissue ferritins was identified, and structural prediction indicated that this motif is located on the surface of the ferritin oligomer. This suggests that the motif may be involved in ferritin transport to radular tissue. In addition, fluorescence was observed in frozen sections of radular tissue from chitons injected with fluorescently labeled hemolymph ferritin. Together, these results suggest that ferritins expressed in specific tissues, secreted into the hemolymph, are transported to radular tissue.