Drafting the CLN3 Protein Interactome in SH-SY5Y Human Neuroblastoma Cells: A Label-free Quantitative Proteomics Approach

Neuronal
ceroid lipofuscinoses (NCL) are the most common inherited
progressive encephalopathies of childhood. One of the most prevalent
forms of NCL, Juvenile neuronal ceroid lipofuscinosis (JNCL) or CLN3
disease (OMIM: 204200), is caused by mutations in the CLN3 gene on chromosome 16p12.1. Despite progress in the NCL field, the
primary function of ceroid-lipofuscinosis neuronal protein 3 (CLN3)
remains elusive. In this study, we aimed to clarify the role of human
CLN3 in the brain by identifying CLN3-associated proteins using a
Tandem Affinity Purification coupled to Mass Spectrometry (TAP-MS)
strategy combined with Significance Analysis of Interactome (SAINT).
Human SH-SY5Y-NTAP-CLN3 stable cells were used to isolate native protein
complexes for subsequent TAP-MS. Bioinformatic analyses of isolated
complexes yielded 58 CLN3 interacting partners (IP) including 42 novel
CLN3 IP, as well as 16 CLN3 high confidence interacting partners (HCIP)
previously identified in another high-throughput study by Behrends
et al., 2010. Moreover, 31 IP of ceroid-lipofuscinosis neuronal protein
5 (CLN5) were identified (18 of which were in common with the CLN3
bait). Our findings support previously suggested involvement of CLN3
in transmembrane transport, lipid homeostasis and neuronal excitability,
as well as link it to G-protein signaling and protein folding/sorting
in the ER.