title: Co-translational processing of glycoprotein 3 from equine arteritis virus: N-glycosylation adjacent to the signal peptide prevents cleavage.
abstract: Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Bioinformatics analyses suggest that a similar processing scheme may exist for some cellular proteins. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, however, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes.
authors.affiliation: From the Institut für Virologie, Free University Berlin, 14163 Berlin, Germany.
authors.forename: Anna Karolina
authors.initials: AK
authors.lastname: Matczuk
authors.forename: Dusan
authors.initials: D
authors.lastname: Kunec
authors.forename: Michael
authors.initials: M
authors.lastname: Veit
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D019295:Computational Biology; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007240:Equartevirus / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046188:Protein Modification, Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: C468294:GP3 protein, equine arteritis virus; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D001216:Asparagine
keywords: Arterivirus; Endoplasmic Reticulum (ER); Glycosylation; Gp3; N-Glycosylation; Protein Translocation; Signal Peptidase; Signal Peptide Cleavage; Virology