Peptide sequence mapping around bisecting glcnac‐bearing n‐glycans in mouse brain

Abstract

N‐glycosylation is essential for many biological processes in mammals. A variety of N‐ glycan structures exist, of which, the formation of bisecting N‐acetylglucosamine (GlcNAc) is catalyzed by N‐acetylglucosaminyltransferase‐III (GnT‐III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc‐modified proteins involved in Alzheimer’s disease and cancer. However, the mechanisms by which GnT‐III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3‐deficient mice. Target glycoproteins of GnT‐III were enriched with E4‐phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph‐mass spectrometry. We identified 32 N‐glycosylation sites (Asn‐Xaa‐Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N‐glycosylation sites that displayed bisecting GlcNAc suggested that GnT‐III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT‐III in the brain, indicated that GnT‐III acts on N‐glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT‐III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped‐back conformation of the modified glycans.