One late infantile variant of the neurodegenerative disease neuronal ceroid lipofuscinosis (NCL) is caused by a mutation in the CLN5 gene. CLN5 encodes a lysosomal glycoprotein whose structure and function have not yet been clearly defined. In the present study, we used epitope-tagged CLN5 to determine the topology and solubility of the CLN5 protein. Our results indicated that CLN5 is synthesized as a type II transmembrane (TM) glycoprotein with a cytoplasmic N-terminus, one TM segment, and a large luminal C-terminal domain containing an amphipathic helix (AH). The cytoplasmic and TM domains were rapidly removed following signal-peptide cleavage, and the resulting mature CLN5 was tightly associated with the lumen of the membrane through the AH. CLN5 pathological mutants deprived of AH lose their membrane association, are retained in the endoplasmic reticulum, and are rapidly degraded by the proteasomal machinery. We experimentally define the topology of CLN5 and demonstrate the existence of an AH that anchors the protein to the membrane. Our work sheds light on the basic properties of CLN5 required to better understand its biological functions and involvement in NCL pathogenesis.
Keywords: CLN5; amphipathic helix; neuronal ceroid lipofuscinoses (NCL); topology; transmembrane domain.
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