Protein glycosylation is an important posttranslational modification that confers both structural and functional properties to the molecules . There are several types of glycosylation with various carbohydrate structures, and biosynthesis of each sugar chain, in general, is an elaborate process of addition and processing of carbohydrates in the endoplasmic reticulum (ER) and Golgi, where a number of glycosyltransferases and glycosidases are involved in these reactions. Glycoprotein degradation, on the other hand, requires many hydrolases to act on carbohydrates and involves intracellular transport of both glycosidases and their substrate glycoproteins. Collectively, enzymes that modulate glycoprotein carbohydrates during their biosynthesis and degradation are called glycoenzymes, which play critical roles in maintaining cellular structure and function. Biosynthesis of carbohydrate moieties of glycoproteins, unlike that of DNA, RNA, or proteins, is a template-independent reaction, that is, their structures are not directly encoded by genes. However, they reflect biochemical reactions catalyzed by glycoenzymes expressed in a cell. In other words, structural integrity of carbohydrates is indirectly determined by a large number of genes coding for glycoenzymes. Therefore, mutations in any of these enzymes that affect the structures and functions of glycoproteins will cause deleterious effects on cellular activities.
Glycosylation is categorized into 2 major classes on the basis of the linkage structure between a polypeptide and a carbohydrate chain, i.e., N-linked and O-linked oligosaccharides (Fig. 1). N-Linked glycans, the most extensively studied class, are the ones attached to an asparagine (Asn) residue in the Asn-X-Ser/Thr triplet sequence (X, any amino acid except proline; Ser, serine; Thr, threonine) in a polypeptide chain. Glycans that are attached to a hydroxylamino acid residue are called O-linked oligosaccharides and can be categorized into several subclasses, according to the types of monosaccharides directly attached to the amino acid (Fig. 1). Among them,
In this article, diseases that are associated with altered protein glycosylation are described. Moreover, to achieve the normal cellular function, glycoenzymes involved in glycosylation processes such as synthesis, processing, and degradation must be under the proper control. Important roles of some of these enzymes are also discussed, with a focus especially on those modulating
2. N-glycan biosynthesis
2.1. Glycoenzymes involved in N-glycan biosynthesis
N-Glycans are characterized by a linkage structure of GlcNAcβ1Asn. Its biosynthesis is distinct from that of other glycans in that a precursor oligosaccharide preassembled on a lipid dolichol (Dol) is
2.2. Disorders in N-glycan biosynthesis: CDG-I and CDG-II
Congenital disorders of glycosylation (CDG), previously known as carbohydrate-deficient glycoprotein syndrome, are a subset of genetic diseases characterized by abnormalities in glycosylation. N-Glycans are physiologically and developmentally important in various tissues, and therefore the complete absence of these oligosaccharides is lethal. Almost all CDGs are autosomal recessive; patients carrying hypomorphic mutant alleles exhibit multiple organ failure, such as neurodevelopmental disorders, hepatopathy, and immunological diseases. Since the biosynthesis of N-glycans consists of 2 distinct processes (i.e., the assembly of precursor oligosaccharides on dolichol and the processing of oligosaccharides after transfer to acceptor proteins), CDGs can be categorized into 2 general types. One type is caused by insufficient assembly of the lipid-linked precursor oligosaccharides in the ER, which is characterized by decreases in the number of N-glycans with normal structures. The other type involves defects in the processing of oligosaccharides that are transferred to proteins, thereby resulting in the production of short and simple sugar chains without affecting the number of glycans. The former type, designated as CDG-I, constitutes defects in the synthesis of dolichol lipid-linked oligosaccharide (LLO) chain as well as its transfer to proteins in the ER. The first report on CDG was made in 1980 , and 15 years later, it was proposed that the disease, CDG-Ia, is caused by the deficiency of an enzyme, phosphomannomutase . This enzyme catalyzes the interconversion of mannose 6-phosphate and mannose 1-phosphate (Fig. 2), and its deficiency leads to a shortage of substrates (GDP-Man and Dol-P-Man) that are required for the synthesis of dolichol-oligosaccharides. CDG-Ia is the most common CDG, and its frequency has been estimated as 1 in 20,000 live births. A less common type of CDG, CDG-1b, possesses deficiency in phosphomannose isomerase (Fig. 2) [6,7]. Without this enzyme, the cells fail to convert fructose 6-phosphate to mannose 6-phosphate and, similarly to CDG-Ia, cannot generate LLOs efficiently. Interestingly, it was found that treating patients with CDG-Ib, but not CDG-Ia, with high-mannose diet alleviates the clinical manifestation, since mannose 6-phospahte can be produced by hexokinase from mannose ingested from food . To date, there are several different types of CDG-I, all of which are due to genetic defects in enzymes involved in the generation of LLOs. CDG-II, on the other hand, involves malfunctions in the trimming or processing of the protein-bound chains in the ER and Golgi. For example, CDG-IIa is caused by mutations in
3. O-glycan biosynthesis
As shown in Fig. 1, there are several subclasses of O-glycosylation, and each subclass has its own biosynthetic process with specific glycosyltransferases. Disorders associated with altered
3.1. Deficiency in O-mannosylation: congenital muscular dystrophies and dystroglycopathies
O-Mannosylation is related to congenital muscular dystrophies with neuronal abnormalities, such as lissencephaly and mental retardation. Muscular dystrophies are genetic diseases with degeneration and disruption of muscle fibers, resulting in the progressive wasting of skeletal muscles and atrophy. The dystrophin-glycoprotein complex (DGC) (Fig. 3) plays a critical role in maintaining muscle integrity [12,13]. It contains αDG, βDG, dystrophin, and other components, and αDG and βDG are produced from a single polypeptide called dystrophin-associated glycoprotein or dystroglycan  after its proteolytic cleavage. Mutations in the DGC components lead to unstable complex formation, which can be a potential cause of the diseases. In fact, dystrophin has been known to be a causative gene of Duchunne-type muscular dystrophy [15,16]. Moreover, among the DGC components, αDG is a peripheral membrane protein with a mucin-like domain, which is heavily modified with O-mannosylglycans. These sugar chains can bind to extracellular matrix proteins, such as laminin, agrin, and perlecan, through their laminin G domains . The reduced αDG glycosylation was observed in a mouse model of muscular dystrophies, myd, indicating that its hypoglycosylation might be a causal factor for the disorders [18,19]. Several studies demonstrated that αDG also contains mucin-type carbohydrates, suggesting that mucin-type O-glycosylation may be involved in the proper glycosylation of αDG . As described below, other mutations also cause αDG hypoglycosylation with a very wide spectrum of phenotypes , and all the diseases caused by the altered αDG glycosylation have been collectively designated as dystroglycanopathies .
O-Mannosylation, a recently appreciated glycosylation, is predominantly observed on αDG. Representative structures of O-mannosylglycans are described in the Fig. 3. The addition of a mannose residue to Ser/Thr in the α-configuration is catalyzed by an enzyme complex composed of protein
It is important to note that only half of congenital muscular dystrophy can be explained by gene mutations described above, suggesting that other alleles also exert effects on the phenotypes [21,22,38]. The Dol-P-mannose synthase subunit DPM3 is one candidate gene; its mutation was found in muscular dystrophy patients whose αDG glycosylation was impaired . Recently, mutations in the
3.2. Deficiency in mucin-type O-glycosylation
3.2.1. Enzymes involved in the biosynthesis of mucin-type O-glycosylation
3.2.2. Involvement of GalNAc-T family in genetic disorders
18.104.22.168. GalNAc-T3 as a modulator of FGF23 levels
Familial tumoral calcinosis (FTC) is a rare, autosomal recessive metabolic disorder that manifests with hyperphosphatemia and massive calcium deposits in periarticular spaces, soft tissues, and sometimes in bone. The gene underlying FTC was mapped to 2q24-q31  which includes the
22.214.171.124. GalNAc-T2 as a regulator of plasma lipid levels
Extensive genomic analyses recently revealed that 1q42 is associated with plasma lipid concentrations [48,49], and the
GalNAc-T17as a candidate gene for Williams–Beuren syndrome and dog domestication
Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder associated with physical, behavioral, and cognitive abnormalities  and is caused by haploinsufficiency of multiple genes at 7q11.23. GalNAc-T17, also known as WBS chromosome region 17/WBSCR17 , was identified in the flanking region (7q11.22) of the chromosomal deletion in WBS patients’ genome. Interestingly, WBSCR17 mRNA is predominantly expressed in the nervous system ; therefore, haploinsufficiency of GalNAc-T17 can be expected to be related to characteristic traits of WBS patients, such as hypersociality and impairment in visuospatial processing. In addition, a recent study on the process of dog domestication reported a single SNP located near WBSCR17 in the dog genome and has implicated WBSCR17 in the early domestication of dogs . It is also noted that GalNAc-T17 is a very inert isozyme, in terms of catalytic activity. GalNAc-T17, together with GalNAc-T8, -T9, and -T18, belongs to the Y subfamily, a group defined by Li
126.96.36.199. GalNAc-Ts in other genetic diseases
Recent genome-wide studies have successively revealed novel loci associated with numerous genetic diseases. Among them, some GALNT genes have been implicated in diverse disorders or tumor susceptibility:
3.2.3. Tn syndrome caused by Cosmc mutations
The Tn syndrome is a rare autoimmune disease. In patients, subpopulations of all blood cell lineages carry an incompletely glycosylated membrane glycoprotein, known as the Tn antigen (GalNAcα1Ser/Thr). Since the anti-Tn antibody is present in most normal human adult sera, patients with Tn syndrome display anemia, leucopenia, and thrombocytopenia . It has been suggested that the Tn syndrome is associated with somatic mutations in the
4. Degradation of carbohydrate units of glycoproteins
4.1. Degradation by lysosomal enzymes
Glycoconjugates, including glycoproteins, glycolipids, and proteoglycans, are biosynthesized intracellularly and incorporated extracellularly by endocytic and phagocytic mechanisms; they are transported via intracellular trafficking to lysosomes, where they are then catabolized . Both N- and O-linked oligosaccharides of glycoproteins are ultimately degraded in lysosomes. Lysosomes contain more than 60 distinct acid hydrolases, so-called lysosomal enzymes, as well as their co-factors, including activators and stabilizing proteins. Most lysosomal glycosidases are classified as exo-type enzymes acting sequentially at the non-reducing termini  and exhibit their optimal catalytic activity under acidic pH conditions because of the function of vacuolar ATPase . Some of them can also localize in non-lysosomal compartments, including early endosomes and cell membrane . In addition, a group of them interact with other enzymes and activator proteins, thereby enhancing the catalytic efficiency through direct binding or presenting the substrates [70-72].
The biosynthesis of lysosomal enzymes and their co-factors is controlled by gene expression , posttranslational modifications, and intracellular trafficking [67,74]. Specifically, soluble matrix enzymes are N-glycosylated in the ER and phosphorylated in the Golgi apparatus at the sixth position of the terminal mannose residues (M6P), via a 2-step reaction catalyzed by Golgi-localized phosphotransferase and uncovering enzyme (
4.2. Lysosomal multienzyme complex for carbohydrate degradation
A group of lysosomal enzymes form a multienzyme complex to regulate their catalytic activities and turnover for efficient catabolism and stabilization. Lysosomal protective protein/cathepsin A (EC188.8.131.52; CTSA) is a multifunctional glycoprotein that exhibits not only catalytic activities but also protective functions [70,75]. CTSA is synthesized as a 452-amino-acid (54-kDa) precursor zymogen that contains intramolecular disulfide bonds and 2 N-glycans [70,75,76]. In the endosomal/lysosomal compartment, the precursor undergoes endoproteolytic processing and is converted to the enzymatically active mature form composed of 32- and 20-kDa subunits [70,75]. The mature enzyme is active at both acidic and neutral pH and functions as cathepsin A (acid carboxypeptidase)/neutral deamidase/esterase on a subset of bioactive peptides, including tachykinins and endothelin-1 [77-79], suggesting its contribution to a variety of intracellular and extracellular cell processes [80,81]. As for the protective functions of CTSA, it forms a multienzyme complex with lysosomal neuraminidase 1 (EC 184.108.40.206; NEU1) and acid β-galactosidase (EC 220.127.116.11; GLB1) to activate NEU1 and protect GLB1 from physiological proteolysis [70,75] The association of NEU1 with CTSA, which probably serves as a molecular chaperone, is particularly crucial for NEU1 neuramindase activity, since the activity is lost in the absence of CTSA, even though GLB1 retains at least 10–15% of the normal enzymatic activity levels [70,75]. The presence of combined deficiency of these enzymes and the excessive accumulation of sialyloligosaccharides derived from glycoproteins reveal the importance and contribution of the multienzyme complex to the physiological degradation of the N- and O-glycans derived from glycoproteins.
5. Disorders of glycoprotein carbohydrates and therapeutic approach
5.1. Lysosomal storage diseases (LSDs) causing metabolic errors of glycoprotein catabolism
Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by genetic defects in the lysosomal enzymes and their cofactors. LSDs result in the excessive accumulation in lysosomes of undegraded substrates, including oligosaccharides derived from glycoproteins, glycosphingolipids, and glycosaminoglycans derived from proteoglycans [66,81]. LSDs comprise more than 40 different disorders, and each incidence is about 1 per 100 thousand births, with differences among disorders and races. The clinical manifestations of LSDs are quite heterogeneous, but many of them involve neurological disorders.
LSDs that are associated with the accumulation of oligosaccharides derived from glycoproteins include α-mannosidosis [82-84], β-mannosidosis [82,83], fucosidosis [82-84], sialidosis [82,85-87], galactosialidosis [70,75], etc. (Table 1). In this chapter, we will focus on sialidosis and galactosialidosis, both of which are associated with NEU1 deficiency and characterized by the accumulation of sialylololigosaccharides and lead to heterogeneous clinical manifestations.
|Sialidosis||Lysosomal sialidase (Neuraminidase 1)||[82,85-87]|
|Schindler disease / Kanzaki disease||α-|||
5.1.1. Sialidosis (MIM 256550)
Sialidosis is an autosomal recessive, lysosomal neuraminidase 1 (
5.1.2. Galactosialidosis (MIM 256540)
Galactosialidosis is an autosomal recessive deficiency in the
5.1.3. Molecular therapy for LSDs
Several therapeutic approaches have been developed and clinically applied to LSDs , including bone marrow transplantation (BMT), enzyme replacement therapy (ERT), gene therapy (GT), and substrate reduction therapy (SRT). BMT, ERT, and
GT has advantages such as a long-lasting treatment with a single administration utilizing recombinant viral gene transfer vectors, [100-103] including retrovirus, adenovirus, herpes simplex virus, adeno-associated virus (AAV), and lentiviruses, albeit with its own attendant concerns, such as low levels and persistence of expression, as well as insertional mutagenesis resulting in neoplasia.
Alternatively, SRT based on prevention of the biosynthesis of natural substrates by utilizing synthetic substrate analogs, has been clinically applied [90,104,105], even though its utility is limited by side effects, continuous administration, and high cost.
In contrast, no definitive treatment is clinically available for LSDs involving glycoprotein catabolism, even though preclinical experiments with animal models have been performed. Heterologous BMT in a ferine α-mannosidosis model caused by a 4-bp deletion in the feline
Moreover, a murine galactosialidosis model (
Several approaches used to develop ERT for sialidosis and galactosialidosis have been challenged. The murine sialidosis model was treated with a short-term, high-dose ERT using baculovirus-derived recombinant
Carbohydrates are very important molecules not only as energy sources but also for a wide variety of cellular functions, ranging from cell-cell interaction to immunity (1). Examples of glycosylation disorders described above clearly demonstrate that addition of carbohydrates to proteins is crucial for maintaining normal cellular physiology and that glycosylation disorders affect various cellular activities. The sugar chains, although not the direct gene products, contain essential information for the proper cellular function. The information, encoded by carbohydrate structures, is regarded as the sugar codes and is deciphered by a collection of carbohydrate-recognizing molecules, such as selectins and galectins [113,114]. In other words, glycosylation disorders can be considered as failures in interpreting the sugar codes. Due to their structural similarity and heterogeneity, carbohydrates are the most difficult molecules to study with the methods commonly used for nucleic acids and proteins. While the structure-function relationship of carbohydrates have remained unclear for a long time, the recent progress in separation, mass spectrometry, and genome-wide association studies of carbohydrates has been bringing information more rapidly than ever[115,116]. We expect that rapidly increasing knowledge of glycan structures and functions will help not only understand the importance of glycosylation in biology and diseases but also, in the near future, exploit the way to treat glycosylation-related diseases.