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Quantitative proteomic analysis of apical interacting partners of canine galectin 8

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Canis Familiaris (dog) (canis Lupus Familiaris)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Chia-Jung Yu

LAB HEAD: Chia-Jung Yu

PROVIDER: PXD038021 | Pride | 2023-11-25

REPOSITORIES: Pride

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Project description:Osteoarthritis is a degenerative joint disease that ranks among the leading causes of pain, adult disability, shortening of working life, and socioeconomic costs worldwide. The mechanisms underlying osteoarthritis pathogenesis are yet to be fully elucidated, thus limiting current disease management and treatment. Galectin-1 is an endogenous carbohydrate-binding protein central to adhesion via glycan-bridging, glycoconjugate-mediated signaling, cell proliferation, differentiation, apoptosis, cancers, and host-pathogen interactions. The chondrocyte glycophenotype, which can act as a system of counter-receptors for galectin binding, is compromised in osteoarthritis. We here investigated Galectin-1 and associated gene network's role in human osteoarthritis pathogenesis. Immunohistochemical analysis showed that Galectin-1 associates with osteoarthritic cartilage and subchondral bone histopathology and severity (p<0.0001, n=29 patients). Glycan-dependent Galectin-1 binding to osteoarthritic chondrocytes' cell surface led to marked upregulation of matrix metalloproteinases and activation of NF-ÎºB. Biochemical, molecular and genome-wide analyses showed that Galectin-1 strongly activates a large inflammatory gene network (p<10-16). Bioinformatic analyses of gene promoters up-regulated by Galectin-1 unveiled an overwhelming NF-ÎºB signaling signature. Inhibition of any of several components of the NF-ÎºB pathway using dedicated inhibitors led to dose-dependent impairment of Galectin-1-mediated transcriptional activation. This study identifies for the first time Galectin-1 as an activator of clinically relevant inflammatory-response genes co-regulated by NF-ÎºB. Since inflammation is critical to cartilage degeneration in osteoarthritis, this report is also first to put glycobiology at the center-stage of osteoarthritic cartilage degeneration. Finally, this is the first report to uncover a Galectin-1 gene signature and associated gene network in any biological setting or species. For microarray experiments, osteoarthritic chondrocytes were isolated from five male patients (47-78 years). Following starvation, cells were incubated in the presence of 50 Âµg/ml recombinant Galectin-1 for 24 h. For each donor population, untreated cells were included as control. In total, 10 samples were analyzed.

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Quantitative proteomic analysis of apical interacting partners of canine galectin 8

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