Translational Glycobiology in Human Health and Disease

Along with nucleic acids, proteins, and lipids, carbohydrates stand as one of four main components of cellular architecture. However, glycobiology (or carbohydrate bioscience) is little understood by non-experts, partly because carbohydrates are a complex, diverse class of molecules structurally and functionally. In recent years, advances in computational analytics (glycomics) have allowed us to better interpret and realize the importance of glycobiology in human health and disease, and glycans and their associated processes have been shown to play a significant role across a variety of disease types. As the biomedical sciences continue to adopt multi-omic and precision medicine approaches, a greater understanding of glycobiology is essential for maintaining healthy physiology and advancing disease treatment.Translational Glycobiology in Human Health and Disease offers a deep examination of glycobiology for experts and non-experts alike in areas ranging from the role of glycobiology in chronic and infectious diseases to advances in technologies for higher throughput analysis and diagnosis. While keeping human health in the forefront, this book integrates a thorough discussion of glycobiology fundamentals with its growing areas of application and societal impact. With emphasis throughout on the interdisciplinary nature of glycosciences, this book also features perspectives from the health, computational (glycoanalytics), materials, biopharmaceutical, and diagnostic sciences.Disease and speciality areas addressed include gycoimmunology, neuroglycobiology, commensal glycobiology, gut health, regenerative medicine and glycobiology, glycobiology and cancer, congenital disorders of glycosylation, infectious disease glycobiology, and parasite glycobiology. Computational approaches discussed, supporting the advance of new research, include advanced glycoanalytics, glycomics microarrays, glycoengineering, and glycol systems biology. Additionally, authors consider impact areas for society and public health, such as glycobiology and entrepreneurship, policy and regulatory requirements for glycosylation, future research, and translation to new diagnostics and drug discovery.

• Provides a deep, foundational overview of glycoscience and its translational potential, highlighting glycobiology’s growing role in human health and disease study
• Examines a broad range of relevant disease areas and applications of glycobiology in policy and public health
• Features chapter contributions from leading, international experts in the field, fully integrating perspectives from the health, computational, materials, biopharmaceutical, and diagnostic sciences

• Language: English
• Publisher: Academic Press
• Release date: Nov 29, 2023
• ISBN: 9780128220023

Book preview

Section 1

Glycobiology

Outline

Chapter 1. Carbohydrates and human glycosylation

Chapter 2. Lectins and their applications in biomedical research

Chapter 3. Carbohydrate-active enzymes

Chapter 4. Carbohydrate sulfotransferases in glycosaminoglycan biosynthesis

Chapter 1: Carbohydrates and human glycosylation

Anup Mammen Oommen, Satbir Kaur Gill, Lokesh Joshi, and Stephen Cunningham     Glycoscience Group, Advanced Glycoscience Research Cluster, University of Galway, Galway, Ireland

Abstract

Glycosylation is one of the most abundant, diverse, and complex forms of posttranslational modification (PTM) shared among nearly all unicellular to multicellular prokaryotic and eukaryotic organisms. The uniqueness of glycosylation PTM stems from the fact that, unlike other forms of PTMs, this phenomenon creates highly dynamic and complex glycan structures depending on the species, the cell type, and the environmental conditions. Involving complex metabolic networks and enzymatic pathways, glycosylation exponentially increases biological complexity and implicates itself in many health and disease functions. It is often regarded as an evolutionary adaptation to bring about a significant expansion in biomolecules without requiring a direct genetic template. Sharing commonalities in glycan synthesis, species-specific glycan structures are generated by the presence of unique sets of enzymes and acceptor substrates across species. However, across organisms, glycosylation plays a crucial role as a structural and functional determinant of the generated glycoconjugates owing to its regulatory control on fundamental aspects of biomolecules such as stability, folding, distribution, and activity. This regulatory control, exerted by the highly dynamic and complex glycoconjugate structures, is spatially organized in both the extracellular and intracellular compartments. This chapter details an overview of the human glycosylation machinery involved in the synthesis and recycling of diverse glycoconjugate structures.

Keywords

Extracellular matrix; Glycan; Glycoconjugate; Glycolipids; Glycosaminoglycan; Glycosylation; N-linked; O-linked; Proteoglycans

1.1. Introduction

The cells of almost all unicellular to multicellular prokaryotic and eukaryotic organisms are coated with a highly diverse carbohydrate envelope [1]. This addition of a carbohydrate coating occurs through glycosylation, a nontemplate-driven posttranslational modification (PTM) process resulting in the synthesis of molecules with a diversity of 104, beyond that of the proteome within humans [2]. From simple sugars (monosaccharides) to complex carbohydrate polymers, the structural versatility of these carbohydrates is highly diverse, dynamic, and complex. This is owing to the structural variation in monosaccharide components, the chemical linkages between the monosaccharides, the anomeric configurations of the individual monosaccharides that can be α or β, and potential branching of the final structures [3]. Synthesis of these structures results from enzyme-driven modification of diverse biological macromolecules like proteins, lipids, and nucleic acids through a process called glycosylation [4,5]. Despite sharing commonalities in their synthesis, species-specific carbohydrate structures are generated by the presence of unique sets of enzymes and acceptor substrates across species.

Unlike the products of the genome and the proteome, carbohydrate structures are not primary gene products and their synthesis occurs without a template. It is estimated that the synthesis of the repertoire of diverse carbohydrate structures is tightly controlled in humans by approximately 700 proteins spatially organized in different cellular components [6,7]. The enzymes involved in the attachment and removal of carbohydrate structures include glycosyltransferases and glycoside hydrolases or glycosidases, carbohydrate-binding proteins (also known as lectins), carbohydrate esterases, glycosaminoglycan- (GAG-)related core proteins, and growth factors, as well as proteins involved in the synthesis and transport of precursor molecules for glycosylation reactions including nucleotide-sugars [7]. Approximately 338 proteins in humans are grouped under 47 glycosyltransferase, 29 glycosidase hydrolase, 1 carbohydrate esterase, and 8 carbohydrate-binding module families in the carbohydrate-active enzymes database (http://www.cazy.org/e355.html), which provides an up-to-date resource for sequence-based family classifications of carbohydrate-active enzymes [8]. The concerted action of these enzymes can generate various forms of the same glycosylated macromolecules, which differ only in their carbohydrate structures, known as glycoforms [9]. Broadly, this includes macroheterogeneity (presence or absence of glycosylation at a particular site of a protein) and microheterogeneity (characterized by different carbohydrate structures on the same site) [4,10]. Different glycoforms have been reported to occur in a tissue-specific manner based on the mass spectrometric analysis of mouse brain and liver glycopeptides [11]. The detailed biological significance of this heterogeneity in glycosylation remains unclear, but it may have originated in parallel with immune system evolution [4]. Certainly, the heterogeneity of carbohydrate structures provides diverse functional adaptations to mammalian physiology. Some well-known examples include roles in cell–cell recognition and interaction, pathogen recognition (and discrimination), fertilization, communication in neuronal tissues, immune system signaling, cell and protein migration, hormonal half-lives in the blood, protein folding and stability, cell development, and embryonic differentiation [12–17]. It is also important to mention lectins, as they mediate the regulatory functions of carbohydrate structures by recognizing specific moieties and thereby communicating the carbohydrate language within the biological system [18].

Several chronic diseases can emanate from any subtle changes in the regulatory control exerted by glycosylation. Currently, more than 100 congenital disorders of glycosylation (CDG) are reported in humans with diverse clinical presentations and symptoms. Most of these diseases have adverse physiological impacts on the skeletal, cardiovascular, and central nervous systems due to genetic mutations in glycosyltransferases, glycosidases, and chaperones, as well as transporter proteins involved in the glycosylation pathways [19]. Similarly, changes in the glycosylation of functional proteins have also been associated with life-debilitating diseases such as diabetes, cancer, autoimmune diseases, and chronic neurodegenerative diseases [20,21]. Despite the challenges in detailed structural characterization and functional association of glycoconjugate structures, technological advancement in the field of glycobiology has significantly benefitted the therapeutic and diagnostic modalities of glycosylation across human disease [22].

In this chapter detailed enzymatic pathways involved in diverse glycosylation processes are explained, focusing on enzymes that are functionally well characterized in mammalian species.

1.2. Glycoconjugate structures

The stereochemistry, linkage position, ring size (furanose or pyranose), and spatial arrangement of 10 basic monosaccharide building blocks [23,24] (Fig. 1.1), constitute the vast array of the human glycome. The collection of N-linked and O-linked oligosaccharides (or glycans) which modify proteins, GAGs which can modify proteins or occur as free polysaccharides, glycolipids, and proteoglycans are known as glycoconjugates [25]. Before synthesis of an oligosaccharide or polysaccharide, monosaccharides are converted into their active high-energy forms known as nucleotide sugars, catalyzed by the nucleotidyltransferase enzymes (EC 2.7.7). This process involves the phosphodiester bond formation between the nucleotide triphosphates, primarily the guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine 5′-triphosphate (CTP) with the phosphorylated form of monosaccharides to form respective nucleoside diphosphate sugars (Table 1.1) [9]. Iduronic acid (IdoA) is formed by the epimerization of glucuronic acid (GlcA), which can impart more conformation flexibility and binding potentials to GAGs [24,26].

Figure 1.1  Matrix depicting the glycosidic bonds that can be formed from 9 active donor sugar nucleotide residues and the 10 basic monosaccharide building blocks. The matrix also captures the glycosidic bonds formed with the amino acid and lipid residues to which they are enzymatically attached. The glycosylation reactions reported for N-, O-, and C-glycosylation are also indicated against the respective amino acid residues. Monosaccharide symbol nomenclature is according to the Consortium for Functional Glycomics.

Specific glycosyltransferase enzymes utilize these active forms of monosaccharides to build oligosaccharides and polysaccharides by forming covalent bonds between them. The glycosidic bond thus formed can involve any hemiacetal hydroxyl group of the donor monosaccharides in the α- or β-configuration to the hydroxyl group of acceptor monosaccharides resulting in both linear and branched chain structures (Fig. 1.1) [27]. In the case of glycoproteins and glycolipids, synthesis of the oligosaccharide structures occurs primarily in the endoplasmic reticulum (ER) and the Golgi components co- and posttranslationally [4,28,29] (Fig. 1.1).

Table 1.1

Key enzymatic reactions involved in the synthesis of activated nucleotide sugar donors essential for the glycosylation pathways. Four out of 10 active nucleotide sugars are synthesized by the conjugation of nucleotides with the four precursor monosaccharides (GlcNAc, Man, Glc, and Neu5Ac) in the first four rows of the table. The remaining active forms of nucleotide sugars are synthesized from these four active nucleotide sugars.CMP, cytidine monophosphate; CTP, cytidine triphosphate; Gal, galactose; GalNAc, N-acetylgalactosamine; GDP, guanosine diphosphate; Glc, glucose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; GTP, guanosine triphosphate; IdoA, iduronic acid; Man, mannose; Neu5Ac, N-acetylneuraminic acid; PO4, phosphate; UDP, uridine diphosphate; UTP, uridine triphosphate; Xyl, xylose.

Similarly, the synthesis of GAGs and proteoglycans is also initiated in the Golgi compartments, except for hyaluronic acid (HA), which occurs primarily at the cell surface [30,31]. Depending on the functional groups of the acceptor molecules on which the glycosidic bonds are formed on proteins, the glycosylation reaction can be broadly classified as N-linked if it is conjugated to the nitrogen atom of asparagine (Asn) amino acid residue (Fig. 1.1) and O-linked if it is linked to the oxygen atom of serine (Ser) or threonine (Thr) amino acid residues on the protein backbone.

1.2.1. Human N-linked glycosylation

Synthesis of the N-linked glycan structure is initiated in the ER by the attachment of an oligosaccharide molecule (comprising 14 monosaccharide units) from a lipid-linked oligosaccharide (LLO) donor, to the amide group of an Asn residue on a nascent polypeptide chain [29] (Fig. 1.2). This highly conserved initiation reaction of the N-glycosylation pathway begins with the conjugation of a monosaccharide on a lipid backbone, which in eukaryotes is based on the longest aliphatic lipid, dolichol phosphate (Dol-P; synthesized de novo from cholesterol precursor molecules) [32]. Biosynthesis of the LLO structure is triggered by the pyrophosphate bond formation with the uridine diphosphate- (UDP-)N-acetylglucosamine (GlcNAc) precursor to form dolichol pyrophosphate GlcNAc (Dol-PP-GlcNAc) [29]. Fourteen steps of sequential addition of sugar moieties, comprising GlcNAc, glucose (Glc), and mannose (Man) residues, result in the formation of LLO structure Glc3Man9GlcNAc2PPDol. The Asn-linked glycosylation (ALG) family of glycosyltransferase enzymes catalyzes the synthesis of LLO across the cytoplasmic and luminal face of ER (Fig. 1.2). Interestingly, the first seven carbohydrate residues of the LLO structure are added directly from the high-energy nucleotide sugars UDP-GlcNAc and guanosine diphosphate- (GDP-) Man, while the next seven are derived from the lipid-linked glycosyl carriers, Dol-P-Man and Dol-P-Glc [33]. The cotranslational attachment of the N-linked glycan core structure is then facilitated in the ER by the multisubunit protein complex oligosaccharyltransferase (OST) to the consensus motif Asn-Xaa-Thr or Asn-Xaa-Thr, where Xaa represents any amino acid except proline (Pro), of newly synthesized protein [34]. Two catalytic complexes of OST are crucial for the N-linked glycosylation pathways in humans [35–37]. Mutations within a number of genes coding for either the catalytic or noncatalytic subunits of OST complexes can result in rare genetic disorders such as congenital disorders of glycosylation-I, X-linked immunodeficiency syndrome, and nonsyndromic autosomal recessive mental retardation [38–40]. Interestingly, the lipid backbones Dol-PP and Dol-P, released during the core N-glycan precursor formation, are recycled back to the cytoplasmic face of ER, which is assumed to be mediated by the flippase proteins [41]. Dol-PP is first dephosphorylated to Dol-P before being recycled into the cytoplasm (Fig. 1.2), which in turn regulates the subsequent formation of lipid-linked glycosyl carriers [42].

Figure 1.2  Overview of the N -linked glycan biosynthetic pathway. Sequence of key enzymatic reactions involved in the de novo core N-linked glycan and subsequent high mannose, complex, and hybrid structure type N-linked glycan synthesis in the ER and golgi organelles. The entire process is categorized into three stages of initiation, processing, and maturation. Initiation: the sequence involved in core N-linked glycan precursor synthesis. Maturation: major glycosylation enzymes involved in directing nascent polypeptide with core N-linked glycan structure to either protein quality control or ERAD pathway. Maturation: the processing of N-glycan on correctly folded proteins. HGNC gene symbols are used to denote the enzymes involved in the synthesis pathways. LLO, lipid-linked oligosaccharide; glucosidase 1 including mannosyl-oligosaccharide glucosidase (MOGS); glucosidase 2 including GANAB, glucosidase II α subunit; OGT complex, O-linked N-acetylglucosamine (GlcNAc) transferase complex; MGAT, mannosyl-glycoprotein N-acetylglucosaminyltransferase. Monosaccharide symbol nomenclature is according to the consortium for Functional glycomics.

Cotranslational modification of nascent polypeptides with core N-linked glycan structures functions as a chemical framework for subsequent glycoprotein processing destined for protein folding, protein degradation, and protein export pathways. This complex ER-dependent protein homeostasis is tightly and precisely controlled by the ER-resident molecular chaperones, exo-glycosidases (mainly glucosidases and mannosidases), glycosyltransferases, and protein disulfide isomerase family members (Fig. 1.2) [43]. In terms of regulation, nascent glycoproteins expressing terminal mono-glucosylated N-linked glycans serve as a retention signal for the ER-resident chaperones to ensure proper folding and subsequent protein secretory pathways by interaction with calnexin and calreticulin in a protein quality control process. CRT and CNX are homologs carbohydrate-binding proteins that interact with glycoproteins only when they have monoglucosylated N-linked oligosaccharides and assist in proper protein folding [44]. Meanwhile, improperly folded glycoproteins displaying terminal α-(1,6)-linked Man on N-linked glycans are withheld in the ER by and directed toward the ER-associated degradation (ERAD) pathways [45] (Fig. 1.2).

Heterogeneity in N-linked glycan structures of properly folded glycoproteins results from a complex series of trimming of the terminal Glc and Man residues by glucosidases and mannosidases, and remodeling by a series of glycosyltransferase enzymes in the Golgi apparatus [46]. The three categories of N-linked glycan structures generated in the Golgi compartments are oligomannose or high mannose type, complex type, and hybrid type (Fig. 1.3) [9].

All of the N-linked glycan structure types share a common pentasaccharide core structure Man5GlcNAc2 (Fig. 1.3). High mannose-type glycans typically contain between five and nine Man residues and the terminal Man is unsubstituted (Fig. 1.3). The activity of the α-1,3-Mannosyl-Glycoprotein 2-β-N-Acetylglucosaminyltransferase (MGAT1) enzyme plays a crucial role in the synthesis of both complex and hybrid type N-linked glycans. By attaching a GlcNAc residue to the α-(1,3)-linked Man of the Man5GlcNAc2 acceptor substrate in the Golgi compartment (Figs. 1.2 and 1.3), it diversifies the subsequent processing steps of the N-linked glycan to complex and hybrid types of N-linked glycan from the high mannose type [49]. Complex type structures have the two terminal Man residues of the core structure elongated by branches formed of monosaccharides other than Man, and Man residues are only present in the core pentasaccharide structure (Fig. 1.3). Golgi-resident glycosyltransferases elongate the oligosaccharide structure with galactose (Gal), GlcNAc, N-acetylneuraminic acid (Neu5Ac) and fucose (Fuc) residues [50,51].

Figure 1.3  Types of N -linked glycan structures. Examples of (A) high mannose, (B) complex, and (C) hybrid N-linked glycan structures. The common pentasaccharide core structure of each N-linked glycan is outlined in a red box. Structures drawn using GlycoWorkbench 2 software [ 47, 48]. Monosaccharide symbol nomenclature is according to the Consortium for Functional Glycomics.

Focusing on mammalian species, modifications of core N-linked glycan structures include fucosylation of the GlcNAc proximal to the protein backbone or the addition of GlcNAc to terminal β-(1,4)-linked Man residues in the core structure [9]. Common additions to the branches of the core structure include the Gal-β-(1,4)-GlcNAc (type 2 N-acetyllactosamine (LacNAc)) or occasionally Gal-β-(1,3)-GlcNAc (type 1 LacNAc) and GalNAc-β-(1,4)-GlcNAc (LacdiNAc) [9]. Synthesis of these structures is primarily regulated by the coordinated activity of certain galactosyltransferases (β-1,4- and β-1,3-galactosyltransferase (B4GALT and B3GALT), glucosaminyltransferases (UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase (B3GNT and N-acetyllactosaminide β-1,6-N-acetylglucosaminyl-transferase (GCNT2)), and galactosaminyltransferases (β-1,4-N-acetyl-galactosaminyltransferase (B4GALNT)). Tandem repeats of type 2 and type 1 LacNAc units generated by B3GNT and B4GALT generate the linear poly-N-acetyllactosamine (poly-LacNAc, or i blood group antigen) [9]. Branching of poly-LacNAc by GCNT2 by adding GlcNAc in a β-(1,6)-linkage yields the I blood group antigen [52]. Similarly, fucosylation of LacNAc and LacdiNAc sequences by the fucosyltransferase-2 (FUT2) and FUT3 enzymes generate the H-antigen blood group and Lewis a (Lea) or Lewis x (Lex) structures, respectively. Further, modification of the H antigen by either the addition of GalNAc or Gal residue to a terminal Gal in an α-(1,3)-linkage results in the generation of A and B antigens [9]. Modification of the Gal residue on Lea and Lex structures by the FUT2 enzyme generates Lewis b (Leb) and Lewis y (Ley) structures [9]. However, if the Gal residue is modified by the ST3 β-galactoside α-2,3-sialyltransferase (ST3GAL) enzymes then it results in the expression of sialyl Lewis a (SLea) and sialyl Lewis x (SLex) structures. Unlike SLea and SLex structures, N-linked glycans with α-(2,6)-linked sialylation display more targeted expression in hepatocytes and leukocytes due to the expression of ST6 β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and play a vital role in immune regulation [53,54]. Interestingly, on certain neuronal proteins like neural cell adhesion molecule (NCAM), polysialic acid (PSA) modifies the terminal glycan structures. PSA is a polymer of α-(2,8)-linked Neu5Ac comprised 50 or more Neu5Ac residues biosynthetically linked by ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 2 and 4 (ST8SIA2, ST8SIA4) [9]. PSA on NCAM modulates axonal guidance during innervation as well as migration of neuronal cells during neural development [9].

Nonsugar modifications such as sulfation by sulfotransferase enzymes also occur among complex N-linked glycans and are a major determinant factor for immune cell migration and homeostasis [55]. Similarly, pathogen evading function was also hypothesized for the sulfated di-, tri-, and tetra-branched N-linked glycans of human Tamm–Horsfall glycoprotein (uromodulin), the most abundant N-linked glycoprotein in human urine [56]. Interestingly, variation from usual complex N-linked glycan structures such as the sulfated LacdiNAc sequence has also been identified to be expressed in human pituitary hormones lutropin and thyrotropin [57].

To date, 27 rare, inherited metabolic disorders have been reported in humans due to defects in N-linked glycosylation pathways [19]. Interestingly, rare mis-sense mutations have also been reported in human species wherein gain in additional consensus site for N-linked glycosylation alters the protein functions and is intrinsically pathogenic [58].

1.2.2. Human O-linked glycosylation

Several O-linked glycosylation types are known. Synthesis of O-linked glycans is initiated by specific enzymes located in the Golgi, ER, and nucleocytoplasmic subcellular compartments by utilizing the seven different types of nucleotide sugars. Categorization of these O-linked glycosylations is based on the primary monosaccharide attached to the proteins and include:

i. O-galactosylation: O-linked Gal to hydroxylysine on the collagen connective tissue protein.

ii. O-glucosylation: O-linked Glc to Tyr residue on glycogenin.

iii. O-xylosylation: O-linked xylose (Xyl) to Ser residue on Notch receptor proteins.

iv. O-mannosylation: O-linked Man to α-dystroglycan.

v. O-fucosylation: O-linked Fuc to Thr residue on tissue plasminogen activator protein [59].

vi. O-GlcNAcylation: O-linked GlcNAc to Ser/Thr residue. Commonly observed on nucleocytoplasmic fraction proteins and is regarded as a key regulatory PTM involved in cell-signaling pathways [60].

Interestingly, some rare glycosylation modifications like the O-linked mannosylation have also been reported to play a vital role in mediating the binding, stabilization, and complex formation between extracellular matrix (ECM) proteins and proteoglycans (such as dystroglycan protein complex with laminin and collagen) [61].

However, mucin-type O-linked glycans are perhaps the most common and best understood of the O-linked glycans and are initiated by the attachment of N-acetylgalactosamine (GalNAc) to the Ser or Thr residues of proteins in the Golgi apparatus [62]. Mucins are heavily O-glycosylated glycoproteins that occur in clusters known as mucin domains found on membrane bound or secreted mucins [63] and are encoded by the MUC family of genes. The single O-GalNAc containing mucin glycan is known as the Tn antigen (GalNAc-α-1-O-Ser/Thr) which is the simplest form generated. Mucin glycans play a vital role in protein structure and stability, modulation of enzyme activity, and have a unique function in providing a hydrated protective outer layer for secretory tissues [64]. Mucin-type oligosaccharides can be divided into a core unit, an elongated chain, and terminating epitopes. Stepwise enzymatic elongation on GalNAc by downstream glycosyltransferases results in the formation of eight core structures (Fig. 1.4), which can be further elongated or modified by sialylation, sulfation, acetylation, fucosylation, and polylactosamine extension [65,66].

Figure 1.4  Mucin-type core structures. Structures drawn using GlycoWorkbench 2 software [47,48]. Monosaccharide symbol nomenclature is according to the Consortium for Functional Glycomics.

The core 1 or T antigen structure is formed by the addition of a Gal residue in a β-(1,3)-linkage to the proximal GalNAc by core 1 β1,3-galactosyltransferase (C1GALT1) (Fig. 1.4). C1GALT1 is the only enzyme that can galactosylate Tn antigen [67]. Both Tn and T antigens can be sialylated on the GalNAc or Gal residues by the ST6 N-acetylgalactosaminide α-2,6-sialyltransferase and the ST3 β-galactoside α-2,3-sialyltransferase enzymes to form the sialyl Tn (STn) and sialyl T (ST) antigens, respectively [67]. The branching of the core 1 structure to form the core 2 structure is effected by the glucosaminyl (N-acetyl) transferase 1, core 2 (GCNT1) enzyme. Addition of GlcNAc-β-(1,3) by B3GNT3 enzyme to the Gal-β-(1,3) residue in core 1 structure limits the substrate availability to GCNT1 enzyme which primarily adds the GlcNAc residue to core 1 structure