Abstract
The N-glycans of mammalian glycoproteins vary greatly in structure, and the biological importance of these variations is mostly unknown. It is widely acknowledged that the bisecting N-acetylglucosamine (GlcNAc) structure, a β1,4-linked GlcNAc attached to the core β-mannose residue, represents a special type of N-glycosylated modification, and it has been reported to be involved in various biological processes, such as cell adhesion, fertilization and fetal development, neuritogenesis, and tumor development. In particular, the occurrence of N-glycans with a bisecting GlcNAc modification on proteins has been proven, with many implications for immune biology. Due to the essential functions of bisecting GlcNAc structures, analytical approaches to this modification are highly required. The traditional approach that has been used for bisecting GlcNAc determinations is based on the lectin recognition of Phaseolus vulgaris erythroagglutinin (PHA-E); however, poor binding specificity hinders the application of this method. With the development of mass spectrometry (MS) with high resolution and improved sensitivity and accuracy, MS-based glycomic analysis has provided precise characterization and quantification for glycosylation modification. In this review, we first provide an overview of the bisecting GlcNAc structure and its biological importance in neurological systems, immune tolerance, immunoglobulin G (IgG), and tumor metastasis and development and then summarize approaches to its determination by MS for performing precise functional studies. This review is valuable for those readers who are interested in the importance of bisecting GlcNAc in cell biology.
Highlights
The monosaccharide-amino acid linkage of N-acetylglucosamine (GlcNAc) β1- asparagine (Asn) was originally discovered in biochemical analyses of abundant glycoproteins present in serum, e.g., immunoglobulins (Imperiali and Hendrickson, 1995; Cobb, 2020)
GlcNAc transferred to the 4-position of the β-linked core mannose (Man) residue in complex or hybrid N-glycans by the β1,4-mannosyl-glycoprotein 4-β-Nacetylglucosaminyltransferase (GlcNAc-T III) is considered as a bisecting structure that is usually not considered as an antenna because it cannot be further extended by the proper enzymes (Narasimhan, 1982; Schachter, 1991; Varki, 2009; Miwa et al, 2012; Chen et al, 2016)
GC was used for the separation of analytes and it has higher resolution for complex small molecules; the glycan samples must be derivatized into partially methylated alditol acetates (PMAA) for GC-mass spectrometry (MS) analysis, and the reaction efficiency affects the quantification of the bisecting GlcNAc structures
Summary
The monosaccharide-amino acid linkage of N-acetylglucosamine (GlcNAc) β1- asparagine (Asn) was originally discovered in biochemical analyses of abundant glycoproteins present in serum, e.g., immunoglobulins (Imperiali and Hendrickson, 1995; Cobb, 2020). The characteristic fragment ions of m/z 118 and 333 were simultaneously detected for the group of 3,4,6-linked Man, which supported the existence of bisecting GlcNAc In this method, GC was used for the separation of analytes and it has higher resolution for complex small molecules; the glycan samples must be derivatized into PMAA for GC-MS analysis, and the reaction efficiency affects the quantification of the bisecting GlcNAc structures. One of these glycoproteins, neprilysin, has the same bisecting GlcNAc location (site N285) as the human neprilysin (P08473) (data not shown) This method can simultaneously obtain precise information regarding the heterogeneity of glycosylation, including the modification sites and their linked glycan structures, which is useful for the functional study of target proteins. Syntheses of glycans or glycoproteins containing bisecting GlcNAc structures have been reported in many papers
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