title: Core-glycosylated mucin-like repeats from MUC1 are an apical targeting signal.
abstract: MUC1 is efficiently delivered to the apical surface of polarized Madin-Darby canine kidney (MDCK) cells by transit through apical recycling endosomes, a route associated with delivery of apical proteins with glycan-dependent targeting signals. However, a role for glycans in MUC1 sorting has not been established. A key feature of MUC1 is a heavily O-glycosylated mucin-like domain with a variable number of nearly perfect tandem repeats and adjacent imperfect repeats. Metabolic labeling, cell surface biotinylation, immobilized lectins, and confocal immunofluorescence microscopy were used to characterize the polarized delivery of MUC1 mutants and chimeras in MDCK cells to identify the apical targeting signal. Both the interleukin-2 receptor α subunit (Tac) and a chimera where the Tac ectodomain replaced that of MUC1 were delivered primarily to the basolateral surface. Attachment of the MUC1 mucin-like domain to the N terminus of Tac enhanced apical but not basolateral delivery when compared with Tac. Conversely, deletions within the mucin-like domain in MUC1 reduced apical but not basolateral delivery when compared with MUC1. In pull-down assays with lectins, we found a notable difference in the presence of core 1 O-glycans, but not poly-N-acetyllactosamine, in apically targeted MUC1 and chimeras when compared with Tac. Consistent with these data, we found no effect on MUC1 targeting when galectin-3, with preference for poly-N-acetyllactosamine, was depleted from polarized MDCK cells. However, we did block the apical targeting activity of the mucin-like repeats when we overexpressed CMP-Neu5Ac:GalNAc-Rα2,6-sialyltransferase-1 to block core O-glycan synthesis. The cumulative data indicate that the core-glycosylated mucin-like repeats of MUC1 constitute an apical targeting signal.
authors.affiliation: Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Carol L
authors.initials: CL
authors.lastname: Kinlough
authors.forename: Paul A
authors.initials: PA
authors.lastname: Poland
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Gendler
authors.forename: Polly E
authors.initials: PE
authors.lastname: Mattila
authors.forename: Di
authors.initials: D
authors.lastname: Mo
authors.forename: Ora A
authors.initials: OA
authors.lastname: Weisz
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D004285:Dogs; D006031:Glycosylation; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D018396:Mucin-1 / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D053645:Interleukin-2 Receptor alpha Subunit; D018396:Mucin-1; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins