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J Biol Chem. 2016 Apr 29;291(18):9835-50. doi: 10.1074/jbc.M115.711838. Epub 2016 Mar 9.

O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking.

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From the Departments of Neuroscience and
the Department of Anatomy, Faculty of Medicine, University of Helsinki, Fin-00014, Helsinki, Finland, and.
the W. M. Keck Foundation Biotechnology Resource Laboratory, Yale/Keck MS and Proteomics Resource, Yale/NIDA Neuroproteomics Center, Yale University, New Haven, Connecticut 06511.
From the Departments of Neuroscience and.
From the Departments of Neuroscience and Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030,


Peptidylglycine α-amidating monooxygenase (PAM) (EC 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.


AtT-20 cell; electron microscopy (EM); furin; lysosome; multivesicular body; neuropeptide; protein secretion

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