Cellular Localization of Debromohymenialdisine and Hymenialdisine in the Marine Sponge Axinella sp. Using a Newly Developed Cell Purification Protocol

Sponges (Porifera), as the best known source of bioactive marine natural products in metazoans, play a significant role in marine drug discovery and development. As sessile filter-feeding animals, a considerable portion of the sponge biomass can be made of endosymbiotic and associated microorganisms. Understanding the cellular origin of targeted bioactive compounds from sponges is therefore important not only for providing chemotaxonomic information but also for defining the bioactive production strategy in terms of sponge aquaculture, cell culture, or fermentation of associated bacteria. The two alkaloids debromohymenialdisine (DBH) and hymenialdisine (HD), which are cyclin-dependent kinase inhibitors with pharmacological activities for treating osteoarthritis and Alzheimer's disease, have been isolated from the sponge Axinella sp. In this study, the cellular localization of these two alkaloids was determined through the quantification of these alkaloids in different cell fractions by high-performance liquid chromatography (HPLC). First, using a differential centrifugation method, the dissociated cells were separated into different groups according to their sizes. The two bioactive alkaloids were mainly found in sponge cells obtained from low-speed centrifugation. Further cell purifications were accomplished by a newly developed multi-step protocol. Four enriched cell fractions (C1, C2, C3, and C4) were obtained and subjected to light and transmission electron microscopy, cytochemical staining, and HPLC quantification. Compared to the low concentrations in other cell fractions, DBH and HD accounted for 10.9% and 6.1%, respectively, of dry weight in the C1 fraction. Using the morphological characteristics and cytochemical staining results, cells in the C1 fraction were speculated to be spherulous cells. This result shows that DBH and HD in Axinella sp. are located in sponge cells and mostly stored in spherulous cells.