Identification of novel N-glycosylation sites at non-canonical protein consensus motifs

Published

June 14, 2016

Author(s)

Mark S. Lowenthal, Kiersta S. Davis, Lisa E. Kilpatrick, Catherine A. Mouchahoir, Karen W. Phinney

Abstract

N-glycosylation is well known to occur at asparagine residues in the canonical consensus sequence N-X-S/T, but has also been identified at a small number of N-X-C motifs including the Asn491 residue of human serotransferrin. Here we report additional novel glycosylation sites within non-canonical consensus motifs, in the conformation N-X-C, based on mass spectrometry analysis of partially-deglycosylated glycopeptide targets. Alpha-1-acid glycoprotein (A1AG) and serotransferrin (Tf) were observed for the first time to be N-glycosylated at a total of six unique non-canonical motifs. N-glycosylation was initially predicted in silico based on the conservation of the N-X-C motif among related mammalian species and demonstrated experimentally in A1AG from porcine, canine, and feline sources and in human serotransferrin. High-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to collect fragmentation data for predicted GlcNAcylated peptides, and to assign modification sites within N-X-C motifs. A combination of targeted analytical techniques that includes complementary mass spectrometry platforms, enzymatic digestions, and partial-deglycosylation procedures, was developed to confirm the novel observations. Additionally, we find that A1AG in porcine and canine sources is highly N-glycosylated at a non-canonical motif (N-Q-C) based on semi-quantitative MRM analysis – the first report of an N-X-C motif exhibiting substantial N-glycosylation. Although reports of non-canonical motif N-glycosylation are uncommon in the literature, this work suggests that it may be more ubiquitous than the current dogma predicts.

Citation

Molecular Biology and Evolution

Volume

15

Issue

7

Pub Type

Journals

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DOI Link

Keywords

glycoprotein, N-glycosylation, consensus motif, mass spectrometry, evolutionary conservation

Citation

Lowenthal, M. , Davis, K. , Kilpatrick, L. , Mouchahoir, C. and Phinney, K. (2016), Identification of novel N-glycosylation sites at non-canonical protein consensus motifs, Molecular Biology and Evolution, [online], https://doi.org/10.1021/acs.jproteome.5b00733 (Accessed October 7, 2025)