title: Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells.
abstract: N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in human embryonic kidney 293 (HEK293) cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.
authors.affiliation: Department of Molecular Pharmacology, RWTH Aachen University of Aachen, Wendlingweg 2, D-52074 Aachen, Germany.
authors.forename: Frauke
authors.initials: F
authors.lastname: Pult
authors.forename: Ghada
authors.initials: G
authors.lastname: Fallah
authors.forename: Ursula
authors.initials: U
authors.lastname: Braam
authors.forename: Silvia
authors.initials: S
authors.lastname: Detro-Dassen
authors.forename: Cristina
authors.initials: C
authors.lastname: Niculescu
authors.forename: Bodo
authors.initials: B
authors.lastname: Laube
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013997:Time Factors; D014162:Transfection; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D001216:Asparagine