Quantitative proteomic analysis of apical interacting partners of canine galectin 8

Primary cilium is a specialized sensory organelle protruding from apical surface of most cells that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials essential for the growing cilia. We demonstrate that the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis, and as the epithelia become polarized, there are more galectin 8 being apically secreted. These extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia, and binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of transition zone. To identify the ciliary molecule which mediates the cilia-lengthening effect of galectin 8, we approached by adopting sequential apical domain selective biotinylation, surface GST-galectin 8 fusion protein incubation, followed by two-step affinity purification scheme. We then processed these apical surface galectin 8-interacting candidates by mass spectrometry. We totally identified 254 reproducible candidates in two independent experiments. There were 96 and 117 plasma membrane candidates noted from the first and second experiments respectively. We focused on 66 candidates categorized as plasma membrane proteins (~25% of total candidates) which were repetitively identified in two separate experiments.