title: O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking.
abstract: Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.
authors.affiliation: From the Departments of Neuroscience and vishwanatha@uchc.edu.
authors.forename: Kurutihalli S
authors.initials: KS
authors.lastname: Vishwanatha
authors.affiliation: the Department of Anatomy, Faculty of Medicine, University of Helsinki, Fin-00014, Helsinki, Finland, and.
authors.forename: Nils
authors.initials: N
authors.lastname: Bäck
authors.affiliation: the W. M. Keck Foundation Biotechnology Resource Laboratory, Yale/Keck MS and Proteomics Resource, Yale/NIDA Neuroproteomics Center, Yale University, New Haven, Connecticut 06511.
authors.forename: TuKiet T
authors.initials: TT
authors.lastname: Lam
authors.affiliation: From the Departments of Neuroscience and.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Mains
authors.affiliation: From the Departments of Neuroscience and Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030, eipper@uchc.edu.
authors.forename: Betty A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D004705:Endocytosis / Q000502:physiology*; D006031:Glycosylation; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D022142:Secretory Vesicles / Q000201:enzymology* / Q000235:genetics
chemical_list: D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase
keywords: AtT-20 cell; electron microscopy (EM); furin; lysosome; multivesicular body; neuropeptide; protein secretion