Abstract
Batten disease or neuronal ceroid lipofuscinoses (NCL) is an inherited lysosomal storage disorder caused by mutated genes referred to as CLNs (ceroid lipofuscinosis neuronal). One such protein is CLN6 (ceroid lipofuscinosis, neuronal, 6), located on the endoplasmic reticulum (ER) membrane. The structure and function of CLN6 is currently unknown. Solving the structure of CLN6 could significantly advance our understanding of its possible substrate interactions, functionality, and role in Batten Disease. To this end, a novel purification method for CLN6 was developed, involving the optimisation of various murine CLN6 constructs, expression systems, detergents, and purification techniques. Additionally, the function of CLN6 was investigated by AlphaFold modelling of publicly available interactome data in search for novel interactions. Furthermore, AlphaFold was used to predict the structure of CLN6 and assess the probability of the proposed CLN6/CLN8 complex, along with other previously proposed partners. Our purification and modelling of CLN6 demonstrates that CLN6 forms a homodimer, supported by the co-purification of two distinct CLN6 constructs. Moreover, the AlphaFold modelling of potential CLN6 interactors showed an enrichment towards membrane proteins of the endoplasmic reticulum-associated protein degradation (ERAD) pathway, one of which, derlin-1 (DERL1), was successfully co-purified with CLN6. These findings suggest a previously unidentified connection between CLN6 and the ERAD pathway or its associated proteins. This study provides a foundation for further investigation into the role of CLN6 in the ERAD pathway, which may elucidate its function and contribution to the pathology of Batten disease, opening new avenues for therapeutic intervention.