N‐glycosylation efficiency is determined by the distance to the C‐terminus and the amino acid preceding an Asn‐Ser‐Thr sequon

Abstract

N-glycosylation is the most common and versatile protein modification. In eukaryotic cells, this modification is catalyzed cotranslationally by the enzyme oligosaccharyltransferase, which targets the b-amide of the asparagine in an Asn-Xaa-Ser/Thr consensus sequon (where Xaa is any amino acid but proline) in nascent proteins as they enter the endoplasmic reticulum. Because modification of the glycosylation acceptor site on membrane proteins occurs in a compartment-specific manner, the presence of glycosylation is used to indicate membrane protein topology. Moreover, glycosylation sites can be added to gain topological information. In this study, we explored the determinants of N-glycosylation with the in vitro transcription/translation of a truncated model protein in the presence of microsomes and surveyed 25,488 glycoproteins, of which 2,533 glycosylation sites had been experimentally validated. We found that glycosylation efficiency was dependent on both the distance to the C-terminus and the nature of the amino acid that preceded the consensus sequon. These findings establish a broadly applicable method for membrane protein tagging in topological studies.

GABRB3 Mutation, G32R, Associated with Childhood Absence Epilepsy Alters α1β3γ2L γ-Aminobutyric Acid Type A (GABAA) Receptor Expression and Channel Gating

“…174 (0.6229), but it slightly reduced the occupancy potential of Asn-33 (␤3 0.5947, ␤3(G32R) 0.5239) (data not shown). Similarly, meta-analyses have concluded that sequons with Arg at the Ϫ1 position are considerably less likely to be glycosylated than sequons with Gly at the Ϫ1 position (32). Finally, although hypoglycosylation disorders frequently cause severe pathology (24), to our knowledge there are no reports of increased glycosylation adversely affecting function or trafficking of other receptors.…”

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

“…24 In our attempts to identify possible helix-helix interactions that stabilize hairpin formation, we focused first on the aspartic residue mentioned above since this appeared to be a likely candidate to promote electrostatic interactions between the TM helices. Because N-glycosylation acceptor sites are absent from the PV 2B sequence, we added a Cterminal glycosylation tag that has been proven to be efficiently modified 25 and synthesized the first 76 residues of the PV 2B (76-mer), which contained the native aspartic residue (Asp74) ( 11), to emphasize the role of this polar residue. In these truncated polypeptides, glycosylation scores translocation of the Ct region across the ER membrane, while non-glycosylated protein bands correspond to insertion of this Ct region (see schemes in Fig.…”

“…44,47 Full-length 2B DNA was amplified from 2B/P2 plasmid using a reverse primer with an optimized Ct glycosylation tag 25 and a stop codon at the end of the 2B sequence (2B-derived expressions). Truncated 2B DNA was amplified by PCR from 2B/P2 plasmid using reverse primers with an optimized Ct glycosylation tag and a stop codon at the end annealing at specific position to obtain the desired polypeptide length (76 residues).…”

Structural Biology of Glycoproteins

“…The occupancy of an N-glycosylation site depends upon the recognition sequon along with the structure of the protein in proximity to the glycosylation site. Some N-glycosylation sites have very low occupancy as a result of the local sequence composition, secondary structure and also distance to the C-terminus [67][68][69]. Bioinformatics programmes such as NetNGlyc and NetOGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/ and http://www.cbs.dtu.dk/services /NetOGlyc/) [70][71] can predict occupancy of N-glycosylation and O-glycosylation sites respectively; however these are sequence based predictions and so need to be verified experimentally.…”