Glycans as complex carbohydrate structures are attached to various other structures, such as lactose, lipids, peptides, proteins, or aminoglycans, present in large amounts in human milk. Glycans play critical roles in many different biological activities. Two common types, N-linked and O-linked glycans, have been extensively analyzed in the last decades. It is essential to identify and quantify O-linked glycans of glycoproteins to determine the changes of glycans. Creative Biolabs has years of experience in glycoengineering studies and we can provide carbohydrate antigen identification of O-linked glycans for clients to promote their projects and accelerate scientific research.
O-linked glycans usually adhere to the peptide chain serine (Ser) or threonine (Thr) residues. O-linked glycoproteins are often large proteins that are normally biantennary with less branching than N-glycans. And contrast to N-linked, O-linked glycosylation is a true post-translational event without a consensus sequence and no oligosaccharide precursor is required for protein transfer. O-linked glycans tend to be very heterogeneous, thus they’re generally classified by their core structures.
Fig.1 Types of O-linked glycans. (Lienemann, 2010)
The most frequent type of O-linked glycans is an initial GalNAc residue (Tn epitope), which is referred to as mucin-type glycans. Other O-linked glycans contain glucosamine, galactose, fucose, mannose, or xylose as the initial sugar bound to Ser/Thr residues. Recently, nonelongated O-GlcNAc groups have been reported to be relevant to phosphorylation states and dynamic processing related to cell signaling in the cell.
O-Linked glycans are prevalent in most secretory cells and tissues. They are found in high concentrations of the zona pelucida surrounding mammalian eggs and may function as sperm receptors. Also, O-linked glycans participate in hematopoiesis, inflammation responses, and the formation of ABO blood antigens.
Glycosylation occurs on side chains of different amino acids and can be categorized based on linkage types between the sugar and amino acid. There’re four glycosylations: N-linked on the amide group of asparagine (Asn); O-linked on hydroxyl groups of serine (Ser), threonine (Thr), hydroxyproline, hydroxylysine, and tyrosine (Tyr); C-linked on tryptophan (Trp), and S-linked on cysteine (Cys). Of them, N- and O-linked are the most known of carbohydrate modifications on the protein in mammals and usual O-linked glycoproteins include glycophorin, mucin, notch, cell fate decisions, thrombospondin, factor VII, factor IX, etc.
The most common type of O-linked glycosylation is the addition of GalNAc to hydroxyls (-OH) of Ser/Thr residues by an O-glycosidic bond, forming the core structure called mucin O-glycans or O-GalNAc glycans. Several other forms of O-linked glycosylation exist, and two of which, O-fucose and O-mannose, have been implicated in protein folding.
Fig.2 Schematic diagram of sequential releases and
analyses of N-linked and O-linked glycans
via the chemoenzymatic method. (Yang, 2017)
It is preferable to analyze both O-glycans and N-glycans from glycoproteins. Technology development to achieve this goal has been the hot spot of glycomics and release of these glycans can be fulfilled by different reactions. O-glycans are released from glycoproteins by chemical methods, while N-glycans are typically released from glycoproteins by enzymes.
O-linked glycosylation takes an important part in biological processes, including protein structure and function. However, the structural elucidation of O-linked glycans remains a major challenge during protein analysis. Today, mass spectrometry (MS) has become a powerful technique for high-quality analytical characterization of O-linked glycans.
Three main MS-based methods are outlined for mucin-type O-glycan analysis: (a) O-glycan profiling (structural analysis of released O-glycan), (b) a "bottom-up" approach (analysis of an O-glycan covalently attached to a glycopeptide), and (c) a "top-down" approach (analysis of a glycan attached to an intact glycoprotein). These MS technical approaches are already major improvements for studying O-linked glycosylation and seem to be valuable for in-depth identification of the type of O-glycan, branching pattern, and the occupancy of O-glycosylation sites.
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