Ceroid lipofuscinosis neuronal 5 (CLN5) is a member of a family of proteins that are linked to neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, known commonly as Batten disease, affects all ages and ethnicities and is currently incurable. The precise function of CLN5, like many of the NCL proteins, remains to be elucidated. In this study, we report the localization, molecular function, and interactome of Cln5, the CLN5 homolog in the social amoeba Dictyostelium discoideum. Residues that are glycosylated in human CLN5 are conserved in the Dictyostelium homolog as are residues that are mutated in patients with CLN5 disease. Dictyostelium Cln5 contains a putative signal peptide for secretion and we show that the protein is secreted during growth and starvation. We also reveal that both Dictyostelium Cln5 and human CLN5 are glycoside hydrolases, providing the first evidence in any system linking a molecular function to CLN5. Finally, immunoprecipitation coupled with mass spectrometry identified 61 proteins that interact with Cln5 in Dictyostelium. Of the 61 proteins, 67% localize to the extracellular space, 28% to intracellular vesicles, and 20% to lysosomes. A GO term enrichment analysis revealed that a majority of the interacting proteins are involved in metabolism, catabolism, proteolysis, and hydrolysis, and include other NCL-like proteins (e.g., Tpp1/Cln2, cathepsin D/Cln10, cathepsin F/Cln13) as well as proteins linked to Cln3 function in Dictyostelium (e.g., AprA, CfaD, CadA). In total, this work reveals a CLN5 homolog in Dictyostelium and further establishes this organism as a complementary model system for studying the functions of proteins linked to NCL in humans.

神经元蜡样质脂褐质沉积症 5 (CLN5) 是与神经元蜡样质脂褐质沉积症 (NCL) 相关的蛋白质家族的成员。这种毁灭性的神经系统疾病通常被称为巴顿病，影响所有年龄段和种族，目前无法治愈。与许多 NCL 蛋白一样，CLN5 的精确功能仍有待阐明。在这项研究中，我们报告了社会性阿米巴盘基网柄菌中 CLN5 同源物 Cln5 的定位、分子功能和相互作用组。人类 CLN5 中糖基化的残基在盘基网柄菌属同源物中是保守的，就像 CLN5 疾病患者中突变的残基一样。盘基网柄菌Cln5 含有假定的分泌信号肽，我们表明该蛋白质在生长和饥饿期间分泌。我们还揭示了盘基网柄菌Cln5 和人类 CLN5 都是糖苷水解酶，这为任何将分子功能与 CLN5 联系起来的系统提供了第一个证据。最后，免疫沉淀结合质谱鉴定出 61 种与盘基网柄菌中的 Cln5 相互作用的蛋白质。在 61 种蛋白质中，67% 定位于细胞外空间，28% 定位于细胞内囊泡，20% 定位于溶酶体。 GO 术语富集分析显示，大多数相互作用蛋白参与代谢、分解代谢、蛋白水解和水解，并包括其他 NCL 样蛋白（例如 Tpp1/Cln2、组织蛋白酶 D/Cln10、组织蛋白酶 F/Cln13）：以及与盘基网柄菌中 Cln3 功能相关的蛋白质（例如 AprA、CfaD、CadA）。 总而言之，这项工作揭示了盘基网柄菌属中的 CLN5 同源物，并进一步将该生物体建立为研究人类 NCL 相关蛋白质功能的补充模型系统。