Identification of Novel N-Glycosylation Sites at Noncanonical Protein Consensus Motifs

详细信息    查看全文

• 作者：Mark S. Lowenthal ; Kiersta S. Davis ; Trina Formolo ; Lisa E. Kilpatrick ; Karen W. Phinney
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：July 1, 2016
• 年：2016
• 卷：15
• 期：7
• 页码：2087-2101
• 全文大小：1531K
• 年卷期：0
• ISSN：1535-3907

文摘

N-glycosylation of proteins is well known to occur at asparagine residues that fall within the canonical consensus sequence N-X-S/T but has also been identified at a small number of asparagine residues within N-X-C motifs, including the N₄₉₁ residue of human serotransferrin. Here we report novel glycosylation sites within noncanonical 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 on asparagine residues within a total of six unique noncanonical motifs. N-glycosylation was initially predicted in silico based on the evolutionary 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 was employed to collect fragmentation data of 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 found that A1AG in porcine and canine sources is highly N-glycosylated at a noncanonical motif (N-Q-C) based on semiquantitative multiple reaction monitoring analysis—the first report of an N-X-C motif exhibiting substantial N-glycosylation. Although reports of N-X-C motif N-glycosylation are relatively uncommon in the literature, this work adds to a growing list of glycoproteins reported with glycosylation at various forms of noncanonical motifs.