Abstract
The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal coimmunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD) but not with the MUC1-ED. Prior pharmacologic inhibition of the NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-butyl amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid, did not diminish NEU1-MUC1-CD association. In addition, glutathione-S-transferase (GST) pull-down assays using the deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 aa of the MUC1-CD. In a cell-free system, we found that the purified NEU1 interacted with the immobilized GST-MUC1-CD and the purified MUC1-CD associated with the immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for the NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either WT NEU1 or a catalytically dead NEU1 G68V mutant diminished the association of the established MUC1-CD binding partner, PI3K, to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.
Highlights
Prior pharmacologic inhibition of NEU1 catalytic activity using the NEU1-selectiveGlycoconjugates expressed on the sialidase inhibitor, C9-BA-DANA, did not surface of all eukaryotic cells contain diminish NEU1-MUC1-cytoplasmic domain (CD) association
Cancer, including intercellular adhesion molecule (ICAM)-1 [24, 25], E-selectin [25], We previously demonstrated that galectin-3 [26], and sialic acid-binding engagement of the MUC1-extracellular domain (ED) with its cognate immunoglobulin-type lectin (Siglec)-9 [27]
A549 cells were pneumonia, and participates in the pathogenesis stimulated with Pa-derived flagellin, lysed, and of cystic fibrosis, bronchiectasis, and chronic the lysates processed for reciprocal obstructive pulmonary disease [29]
Summary
Prior pharmacologic inhibition of NEU1 catalytic activity using the NEU1-selectiveGlycoconjugates expressed on the sialidase inhibitor, C9-BA-DANA, did not surface of all eukaryotic cells contain diminish NEU1-MUC1-CD association. To further support that NEU1 did not associate with the MUC1-ED, binding assays were performed using the MUC1-ED protein containing an NH2-terminal 6XHis epitope tag (Fig. 2G) immobilized on nickelnitrilotriacetic acid (Ni-NTA)-agarose. The purified, unbound MUC1-ED-WT and deletion mutants were incubated with the validated GSTNEU1 immobilized on glutathione-agarose beads and the NEU1-binding proteins processed for 6XHis (MUC1-ED) immunoblotting (Supplemental Fig. S2D, lanes 1-4, 6-8).
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