Glycosaminoglycans (GAGs) are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs.

1. Introduction

Glycosaminoglycans (GAGs) are covalently attached to the core proteins that form proteoglycans (PGs), which are ubiquitously distributed in extracellular matrix and on the cell surface [17]. GAGs are linear polysaccharides that form the side chains of PGs and have been classified into chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin based on their structural units. The backbone of CS consists of repeating disaccharide units of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcUA) (Figure 1). DS is a stereoisomer of CS and composed of GalNAc and L-iduronic acid (IdoUA) instead of GlcUA (Figure 1). They are often distributed as CS-DS hybrid chains in mammalian tissues [8]. On the other hand, HS and heparin consist of N-acetyl-D-glucosamine (GlcNAc) and GlcUA or IdoUA (Figure 1). The glucosamine (GlcN) residues in HS and heparin are modified by not only N-acetylation but also N-sulfation. These GAG chains are modified by sulfation at various hydroxy group positions and also by the epimerization of uronic acid residues during the biosynthetic process, thereby giving rise to structural diversity, which plays an important role in a wide range of biological roles including cell proliferation, tissue morphogenesis, infections by viruses, and interactions with various growth factors, cytokines, and morphogens [718].

Glycosyltransferases, epimerases, sulfotransferases, and related enzymes in the biosynthesis of GAGs have been cloned and characterized (Tables 14 and Figures 2 and 3) [6, 7, 14, 19]. Furthermore, genetic analyses using model animals including mice, zebrafish, fruit flies, and nematodes have led to new findings on different phenotypes [4, 8, 9, 12, 13]. Human genetic disorders including bone and skin diseases caused by mutations in the genes encoding the biosynthetic enzymes for GAGs have recently been reported [7, 14, 20]. This review focused on recent advances in knockout mice for GAG biosynthesis, as well as cartilage and connective tissue disorders caused by disturbances in the biosynthesis of functional GAG chains.

2. Biosynthesis of 3′-Phosphoadenosine 5′-Phosphosulfate

The sulfation of GAGs is required for the exertion of their physiological functions. Sulfotransferases catalyze the transfer of sulfate from the donor substrate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), to the corresponding acceptor substrates [21]. PAPS is synthesized from ATP and inorganic sulfate in the cytosol, and the reaction takes place in two sequential steps [2123]. ATP sulfurylase firstly catalyzes the reaction between ATP and inorganic sulfate to form the biosynthetic intermediate, adenosine 5′-phosphosulfate (APS) [22, 23]. The formation of the active sulfate, PAPS, is then catalyzed by APS kinase, which involves a reaction between APS and ATP [22, 23]. ATP sulfurylase and APS kinase are encoded by the respective genes in bacteria, fungi, yeast, and plants [21]. On the other hand, both enzymes are fused in animals, resulting in a polypeptide designated PAPS synthase (PAPSS), which is a bifunctional enzyme composed of the N-terminal APS kinase domain and C-terminal ATP sulfurylase domain [21]. Following the formation of PAPS in the cytosol, PAPS is translocated into the Golgi by PAPS transporters [24].

3. Biosynthesis of GAG Chains

3.1. GAG-Protein Linkage Region

CS, DS, HS, and heparin chains are attached to serine residues in core proteins through the common GAG-protein linkage region tetrasaccharide, GlcUAβ1-3galactoseβ1-3galactoseβ1-4xyloseβ1-O- (GlcUA-Gal-Gal-Xyl-O-) (Figure 2) [1, 5]. The transfer of a Xyl residue from uridine diphosphate (UDP)-Xyl to specific serine residues in the newly synthesized core proteins of PGs in the endoplasmic reticulum and cis-Golgi compartments is initiated by β-xylosyltransferase (XylT) (Figure 2 and Table 2) [25, 26]. β1,4-Galactosyltransferase-I (GalT-I), which is encoded by B4GALT7, then transfers a Gal residue from UDP-Gal to the Xyl-O-serine in the core proteins [27, 28]. β1,3-Galactosyltransferase-II (GalT-II), which is encoded by B3GALT6, transfers another Gal residue from UDP-Gal to the Gal-Xyl-O-serine [29]. Finally, β1,3-glucuronosyltransferase-I (GlcAT-I), which is encoded by B3GAT3, transfers a GlcUA residue from UDP-GlcUA to the Gal-Gal-Xyl-O-serine (Figure 2 and Table 2) [30]. These enzymes may form a multienzyme complex such as the so-called GAGosome for GAG synthesizing enzymes for the construction of the linkage region [31, 32].

Several modifications including the 2-O-phosphorylation of the Xyl residue as well as sulfation at the C-6 position of the first Gal and at C-4 or C-6 of the second Gal residue have been reported [5]. GAG-Xyl kinase, encoded by FAM20B, Xyl phosphatase, encoded by ACPL2, and Gal-6-O-sulfotransferase, encoded by CHST3 (C6ST1), have so far been identified (Table 2) [3335]. These modifications affect the glycosyltransferase reactions of GalT-I and GlcAT-I in vitro and may regulate the formation of GAG chains [36, 37].

3.2. Repeating Disaccharide Region of CS and DS

Chain polymerization of the repeating disaccharide region in CS and DS chains is initiated by the transfer of the first GalNAc from UDP-GalNAc to the GlcUA residue in the linkage region tetrasaccharide, GlcUA-Gal-Gal-Xyl-O-, by β1,4-N-acetylgalactosaminyltransferase-I (GalNAcT-I) (Figure 2) [193196]. Alternatively, the transfer of a GlcNAc residue from UDP-GlcNAc to the linkage region tetrasaccharide by α1,4-N-acetylglucosaminyltransferase-I (GlcNAcT-I) is known to result in the initiation of the repeating disaccharide region of HS and heparin chains (Figure 2) [197201]. Six chondroitin synthase family members have been identified including chondroitin synthases (ChSys), chondroitin-polymerizing factor (ChPF), and CSGalNAcTs (Figure 2 and Table 3) [193196, 202208]. ChSy1 is composed of 802 amino acids and is a bifunctional glycosyltransferase that exhibits CS-GlcAT-II and GalNAcT-II activities, which are required for the biosynthesis of the repeating disaccharide region, -4GlcUAβ1-3GalNAcβ1 (Table 3) [202]. ChSy1 itself is unable to construct the backbone of CS by the activity of polymerase, whereas the enzyme complex of ChSy with ChPF can form the repeating disaccharide region [203205]. A precursor of CS, the chondroitin backbone, is then maturated by sulfation modified by various sulfotransferases such as uronosyl 2-O-sulfotransferase (UST) [209], chondroitin 4-O-sulfotransferases (C4ST) [210212], chondroitin 6-O-sulfotransferase (C6ST) [213, 214], and GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) [215] (Figure 3 and Table 3). These transfer the sulfate group from the sulfate donor PAPS to the corresponding position of the GlcUA and GalNAc residues in chondroitin. C4STs have been shown to regulate the chain length and amount of CS coordinating with CSGalNAcTs [216, 217].

Epimerization of the C-5 position of GlcUA residues in a chondroitin polymer as a precursor backbone occurs during or after the chain elongation, which results in the formation of the repeating disaccharide region, -4IdoUAα1-3GalNAcβ1-, of DS chains (Figure 3) [218221]. The dermatan chains fully develop through sulfation catalyzed by dermatan 4-O-sulfotransferases (D4ST) [222, 223] and uronosyl 2-O-sulfotransferase (UST) [209] (Figure 3 and Table 3).

3.3. Repeating Disaccharide Region of HS and Heparin

Following the construction of the linkage region tetrasaccharide, GlcUAβ1-3Galβ1-3Galβ1-4Xylβ1-O-serine, on the core protein, transfer of the GlcNAc residue from UDP-GlcNAc to the tetrasaccharide induces chain polymerization of the repeating disaccharide region of HS and heparin catalyzed by GlcNAcT-I [197201] (Figure 2). After the addition of the first GlcNAc to the linkage region, the growing pentasaccharide is further elongated by alternate additions of GlcUA and GlcNAc from UDP-GlcUA and UDP-GlcNAc by HS-β1,4glucuronyltransferase-II (HS-GlcAT-II) and α1,4-N-acetylglucosaminyltransferase-II (GlcNAcT-II), respectively (Figure 2). Exostosin 1 (EXT1) as well as 2 (EXT2) both exhibit HS-GlcAT-II and GlcNAcT-II activities [199, 224226] (Table 4). Furthermore, the heterodimeric complex of EXT1 and EXT2 exhibits HS polymerase activity on a linkage region tetrasaccharide acceptor in vitro, which results in the biosynthesis of HS and heparin polysaccharides [227, 228]. Three homologous genes to the EXT have been identified [6, 14, 229]. EXTL1 and EXTL2 exhibit GlcNAcT-II and GlcNAcT-I activities, respectively, whereas EXTL3 has not only GlcNAcT-I, but also GlcNAcT-II activities (Figure 2 and Table 4) [200, 201].

After the formation of the repeating disaccharide backbone of HS chains by EXTs and EXTLs, GlcNAc residues are converted into GlcN residues by GlcNAc N-deacetylase (Figure 3) [6, 14, 198]. A sulfate group is subsequently transferred from PAPS to the GlcN by GlcN N-sulfotransferase [6, 14, 198]. Both enzymes are encoded by a single gene, GlcNAc N-deacetylase/N-sulfotransferase (Figure 3 and Table 4) [230233]. The interconversion of GlcUA to IdoUA in HS and heparin is achieved by HS-glucuronyl C5-epimerase (Figure 3) [234236]. Moreover, sulfation at the C-2 position of uronic acid as well as C-3 and C-6 positions of the GlcN residues in the HS and heparin are catalyzed by HS 2-O-sulfotransferase, HS 3-O-sulfotransferase, and HS 6-O-sulfotransferase, respectively (Figure 3 and Table 4) [237244]. The desulfation of 6-O-sulfated GlcNS residues in HS chains by HS 6-O-endosulfatase modifies the fine structure of HS in order to regulate various biological events including cell signaling, tumor growth, and angiogenesis (Figure 3 and Table 4) [245247].

4. Knockout and Transgenic Mice of GAG Biosynthetic Enzymes

4.1. Xylt1

A recessive dwarf mouse mutant (pug) obtained from an N-ethyl-N-nitrosourea mutagenesis screen was attributed to a missense mutation in Xylt1, which resulted in the substitution of an amino acid (p.Trp932Arg) [59]. XylT activity in the pug mutant was markedly reduced in vitro, which resulted in a decrease in the amount of GAGs in cartilage. Furthermore, early ossification was reported in this mutant, which resulted in a shorter body length than that of a wild-type embryo. These phenotypes may be caused by an upregulation of Indian hedgehog signaling but not MAPK signaling due to lack of GAGs [59].

4.2. B3gat3 (GlcAT-I)

Mice deficient in GlcAT-I synthesize a smaller CS and HS chain in their blastocysts than that of the heterozygous mice [75]. In addition, these mice exhibit an embryonic lethality before the 8-cell stage due to the failure of cytokinesis, which has been attributed to a deficiency in CS, but not HS based on the findings reported in embryos treated with chondroitinase and heparinase [76]. Moreover, interaction of CS with E-cadherin, which regulates the differentiation of embryonic stem cells, may control Rho signaling pathway [76]. These findings indicated that CS, but not HS, is involved in regulating cell division in mammals.

4.3. Csgalnact1 and Csgalnact2

CSGalNAcT1-null mice have been shown to produce a smaller amount as well as a shorter length of CS chains than the wild-type [84, 85]. These mice also have shorter limbs and axial skeleton and a thinner growth plate in cartilage than wild-type mice, which results in a slightly shorter body length and smaller body weight [84, 85]. It is likely that the reduction in CS may affect normal chondrogenesis and formation of type II collagen fibers [84]. These findings suggest that CSGalNAcT1 is essential for the differentiation and maturation of cartilage.

A deficiency in CSGalNAcT1, but not CSGalNAcT2, has been shown to promote axonal regeneration following spinal cord injury [86]. CS-PGs function as barrier-forming molecules during axonal regeneration after damage to the nervous system [10]. Thus, the down- and upregulation of CS and HS biosynthesis, respectively, in the scars of mice led to better recovery from injuries in the nervous system than the wild type.

4.4. Chsy1

Chsy1-deficient mice are viable but exhibit chondrodysplasia, progression of the bifurcation of digits, delayed endochondral ossification, and reduced bone density [81]. Furthermore, a decrease in 4-O-sulfation and increase in 6-O-sulfation as well as desulfation of the GalNAc residues of CS have been reported in the cartilage of Chsy1−/− mice. The signaling of hedgehog but not of FGF, bone morphogenetic protein, or transforming growth factor-β altered in primary chondrocytes from Chsy1-deficient mice [81], which suggests that CS-PGs and hedgehog protein may coordinately regulate skeletal development and digit patterning.

4.5. Chpf

Mice deficient in Chpf, also known as chondroitin sulfate synthase-2 (CSS2), are fertile and viable and exhibit no obvious abnormalities including osteoarthritis and cartilage development [82]. These findings are consistent with the study by Wilson et al. [81].

4.6. Dse and Dsel

The body weight of Dse−/− mutant mice, which have fewer IdoUA residues in the skin, is ~30% smaller than that of the wild type [88, 89]. Although no significant differences were observed in the content of collagen between Dse−/− and the wild type, the ultrastructure of collagen fibrils in the dermis and hypodermis was thicker in the deficient mice than in the wild type, and a decline in their mechanical strength was also noted in the deficient mice. On the other hand, no morphological or histological abnormalities have been reported in mice targeted with the disruption of DS epimerase-2 encoded by Dsel [93]. In addition, 4-O-sulfation of the DS chain was decreased in the brain of Dse2−/−, whereas the adult Dse2−/− brain had normal structures in the extracellular matrix. The function of Dse2 appears to be compensated by Dse1 [93].

4.7. Chst3 (C6st1)

The number of 6-O-sulfated disaccharide units including the C-unit (GlcUA–GalNAc6-O-sulfate) and D-unit (GlcUA2-O-sulfate–GalNAc6-O-sulfate) was shown to be markedly reduced in the spleens and brains of C6st1-deficient mice, and the number of naive T lymphocytes was also decreased in the spleen [114]. However, brain development in C6st1−/− mice is normal in spite of a decrease in D-units in the CS chains of the null mice.

CS-PGs are newly synthesized in the central nervous system following injury, and this inhibits axonal regeneration [10, 248]. Furthermore, upregulation of the expression of C6st1 and 6-O-sulfated CS-PGs has been demonstrated in glial scars after a cortical injury [249]. C6st1−/− mice had fewer or a similar number of regenerative axons after axotomy to the wild type [115].

An increase in chondroitin 6-O-sulfation was observed in the developing brains of C6st1-transgenic mice and affected the formation of the perineuronal nets and cortical plasticity [116], which are specialized structures of the dense organized matrix, which are composed of CS-PGs, hyaluronan, tenascins, and link proteins and regulate neuronal plasticity and neuroprotection [250]. Chondroitin 6-O-sulfate may regulate the maturation of parvalbumin-expressing interneurons through the incorporation of Otx2 [116], which regulates ocular dominance plasticity.

4.8. Chst11 (C4st1)

The C4st1 gene was identified as a target gene of bone morphogenetic protein signaling using gene trap experiments [94]. C4st1-mutant mice exhibit severe dwarfism and die within six hours of birth due to respiratory failure [95]. Moreover, severe chondrodysplasia with abnormalities in the cartilage growth plate and chondrocyte columns, marked reductions in GAG content and 4-O-sulfated CS, the downregulation of bone morphogenetic protein signaling, and the upregulation of transforming growth factor-β have been observed in these mice. These findings indicated that C4ST1 and the 4-O-sulfation of CS chains were essential for the signaling pathways of bone morphogenetic protein and transforming growth factor-β as well as cartilage morphogenesis.

4.9. Chst14 (D4st1)

D4st1−/− mice have a smaller body weight, a kinked tail, and more fragile skin and are less fertile than the wild type. [107]. In addition, axonal regrowth is initially facilitated in D4st1−/− mice following nerve transection.

Furthermore, the impaired proliferation of neural stem cells, reduced neurogenesis, and an altered subpopulations of radial glial cells have been reported in D4st1-deficient mice [96]. The epitope structure recognized by the monoclonal anti-CS antibody 473HD, which contains the D-unit (GlcUA-2-O-sulfate–GalNAc-6-O-sulfate) and iA-unit (IdoUA–GalNAc4-O-sulfate) in the CS-DS hybrid chains on PGs, such as phosphacan, is required for the formation of neurospheres and as a marker for radial glial cells [251]. Expression of the 473HD epitope was shown to be decreased in the neural stem cells of D4st1−/− mice, and this resulted in the altered formation of neurospheres [96]. These findings indicated that DS chains and/or D4ST1 are essential for the proliferation and differentiation of neural stem cells.

4.10. Chst15 (Galnac4s-6st)

Galnac4s-6st-null mice are viable and fertile and completely defective in the E-unit, GlcUA-GalNAc(4-,6-O-disulfates), in both CS and DS chains [117]. The activities of carboxypeptidase A and tryptase from bone marrow-derived mast cells in Galnac4s-6st−/− were lower than those in the wild type, which suggested that the E-unit-containing CS chain or CS-PGs may be involved in the retention of these proteases in the granules of mast cells.

4.11. Ext1 and Ext2

Gene knockout mice produced by the targeted disruption of the gene encoding Ext1 and Ext2 died by embryonic day 8.5–14.5 due to defects in the formation of the mesoderm and a failure in egg cylinder elongation [119121, 136]. The GlcUA and GlcNAc transferase activities are decreased and HS chains are shorter in mice carrying a hypomorphic mutation in EXT1 generated by gene trapping, which affect the signaling pathways of Indian hedgehog and parathyroid hormone-related peptide [120, 121]. Thus, it is difficult to analyze the in vivo functions of HS chains using conventional knockout mice. A growing number of conditional knockout mice produced by targeted disruption of the gene encoding HS biosynthetic enzymes has provided an insight into the physiological functions of HS and HS-PGs [14]. For example, pluripotent embryonic stem cells in which Ext1 was disrupted fail to differentiate into neural precursor cells and mesoderm cells due to the enhancement of Fgf signaling and retention of the high expression of Nanog [122, 123]. Conditional Ext1-knockout mice selectively disrupted in the nervous system die within the first day of life and have defective olfactory bulbs, midbrain-hindbrain region, and axon guidance due to a disturbance in signaling pathways including Fgf8 and Netrin-1 [124126]. Conditional Ext1-knockout mice specific for postnatal neurons exhibit a large number of autism-like phenotypes in spite of a normal morphology in the brain [127]. On the other hand, mice in which Ext1 was specifically disrupted for chondrocytes and the limb bud, Ext2 heterozygous mice, and compound Ext1+/−/Ext2+/− mice display severe skeletal defects with cartilage differentiation and chondrocyte maturation, and these defects resembled an autosomally dominant inherited genetic disorder, human hereditary multiple exostoses [128132]. Disruption of the Ext1 gene in glomerular podocytes results in an abnormal morphology in these cells [133]. Furthermore, conditional knockout mice lacking Ext1 in the high endothelial venules and vascular endothelium cells show a decrease in lymphocyte homing to peripheral lymph nodes and a compromised contact hypersensitivity response [134, 135]. These findings suggest that HS and HS-PGs are essential for playing a role in their physiological functions in a tissue-specific manner.

4.12. Extl2 and Extl3

Mice deficient in Extl2 are viable and develop normally; however, they produce a larger amount of GAG chains [137, 138]. Liver regeneration was shown to be impaired in these knockout mice following liver injury induced by administration of CCl4 due to suppression of the response to hepatocyte growth factor [137].

Mice deficient in Extl3 are embryonically lethal, which is similar to mice lacking Ext1 or Ext2 [139]. In addition, selective inactivation of the Extl3 gene in pancreatic islet β-cells caused an abnormal morphology as well as a reduction in the proliferation of the islets, which resulted in defective insulin secretion [139]. However, it remains to be determined how HS, HS-PGs, or Extl3 is involved in insulin secretion.

4.13. Ndst1, 2, and 3

Functional analyses of HS and heparin using Ndst1-deficient mice have been performed in approximately 20 studies to date [140164]. Representative studies have been reviewed in this chapter. Ndst1-deficient mice die after birth and have cerebral hypoplasia, axon guidance errors, defects in the eye and olfactory bulbs, insufficient milk production caused by a defect in lobuloalveolar expansion in the mammary gland, and morphological abnormalities in the podocytes [140142, 145, 156, 157, 162, 164]. Ndst1 conditional knockout mice specific for the liver accumulated triglyceride-rich lipoproteins due to a reduction in the clearance of cholesterol-rich lipoprotein particles [148, 163]. Furthermore, mice with the endothelial-targeted deletion of Ndst1 exhibited suppressed experimental tumor growth and angiogenesis including microvascular density and branching of the surrounding tumors due to altered responses to Fgf2 and Vegf, which resulted in reduced Erk phosphorylation [147] and attenuated allergic airway inflammation [151].

Embryos from Ndst2-deficient mouse are viable and fertile, whereas their mast cells are unable to synthesize heparin, which leads to changes in morphology and severely reduced amounts of granule proteases [165167]. These findings indicated that the storage of proteases in granules is controlled by heparin or heparin-PG, such as serglycin [165167]. On the other hand, Ndst3-deficient mice develop normally and are fertile [168].

4.14. Glce (HS GlcUA C5-epimerase)

Mice with the targeted disruption of HS epimerase die immediately after birth and have agenesis of the kidney, a shorter body length, and lung defects [169, 170]. Furthermore, developmental abnormalities in the lymphoid organs, including the spleen, thymus, and lymph nodes, have been reported in the knockout mice [171, 172]. IdoUA-containing HS chains are critical for early morphogenesis of the thymus through binding with Fgf2, Fgf10, and bone morphogenetic protein 4 [171]. In addition, the interaction of HS with a proliferation inducing ligand, hepatocyte growth factor, and CXCL12α is required for B-cell maturation [172].

4.15. Hs2st

Gene trap mice lacking Hs2st die during the neonatal period and exhibit renal aplasia and defects in the eyes, skeleton, and retinal axon guidance [173179]. In addition, the cell-specific disruption of Hs2st in the endothelial and myeloid cells enhanced the infiltration of neutrophils due to an increase in their binding to IL-8 and macrophage inflammatory protein-2 [162]. Mice with the specific disruption of Hs2st in the liver accumulate plasma triglycerides and the uptake of very-low-density lipoproteins is reduced, whereas mice with the specific disruption of Hs6st in the liver do not. These findings suggest that the clearance of plasma lipoproteins is dependent on the 2-O-sulfation of HS [153].

4.16. Hs3st1

HS3ST1−/− mice display normal development and anticoagulant activity [184]; however, it was previously demonstrated that the GlcN 3-O-sulfate structure was essential for the anticoagulant activity of heparin and HS [252]. Other HS3ST family members such as HS3ST2, HS3ST3a, HS3ST3b, HS3ST4, HS3ST5, and HS3ST6 may compensate for the loss of HS3ST1 [184].

4.17. Hs6st1 and Hs6st2

HS6ST1-null mice die during the late embryonic stage, are smaller than the wild type at birth, and have defective retinal axon guidance due to the disturbance of Slit-Robo signaling [177, 178, 181]. In contrast, HS6ST2-deficient mice develop normally [183]. However, serum levels of thyroid-stimulating hormone and the thyroid hormone, thyroxin, are higher and lower, respectively, in the deficient mice, which cause a reduction in energy metabolism with an increase in body weight [183]. The storage of mast cell proteases is altered in double knockout mice with HS6ST1−/−/HS6ST2−/− [182], and their embryonic fibroblasts are partially defective in FGF signaling [253].

4.18. Sulf1 and Sulf2 (HS 6-O-endosulfatase)

Sulf1−/− mice exhibit no apparent abnormalities [185]. On the other hand, Sulf2−/− mice have a smaller body size and mass [185, 192]. Mice deficient in both Sulf1 and Sulf2 have multiple defects including skeletal and renal malformations, which result in neonatal lethality [186]. HS 6-O-sulfation and/or desulfation by Sulfs are known to be involved in the cartilage homeostasis mediated by bone morphogenetic protein and Fgf [187], dentinogenesis through Wnt signaling [188], neurite outgrowth mediated by glial cell line-derived neurotrophic factor [189], muscle regeneration [190], and brain development [191]. These findings indicate that the fine-tuning of 6-O-sulfation by Sulfs may control multiple functions of HS chains during morphogenesis.

5. Human Disorders Affecting the Skeleton and Skin due to the Disturbance of GAGs

5.1. PAPSS2

Spondyloepimetaphyseal dysplasia of Pakistani type, which is characterized by kyphoscoliosis, generalized brachydactyly, short and bowed lower limbs, and enlarged knee joints, is caused by mutations in PAPSS2: p.Ser438X and p.Arg329X [43, 44].

Patients with mutations in PAPSS2, resulting in the substitution of corresponding amino acids (p.Thr48Arg, p.Arg329X, and p.Ser475X), also have spondylodysplasia and premature pubarche, which are accompanied by a short stature, bone dysplasia, excess androgens, hyperandrogenic anovulation, and the loss of dehydroepiandrosterone sulfate [45]. Sulfotransferase 2A1 has been shown to transfer a sulfate group from PAPS to dehydroepiandrosterone (DHEA) in the adrenal glands and liver, resulting in the formation of DHEA-sulfate [254]. The inactivation of PAPSS2 inhibits of not only the formation of PAPS but also the conversion of DHEA into DHEA-sulfate, which leads to the accumulation of DHEA in patients [45]. Excess DHEA is finally converted to testosterone through androsterone.

Autosomal recessive brachyolmia, which is a heterogeneous group of skeletal dysplasias and primarily affects the spine, is also caused by PAPSS2 mutations [46, 47]. Brachyolmia is characterized by a short stature due to a short trunk, irregular endoplates, a narrow intervertebral disc, calcification of cartilage in the ribs, a short femoral neck and metacarpals, and normal intelligence [4648]. However, the excess amount of androgens cannot be detected in these patients. Furthermore, PAPS synthase activity was absent in the recombinant mutant enzymes, including p.Cys43Tyr, p.Leu76Gln, and p.Val540Asp [47].

5.2. XYLT1

Mutation in XYLT1 causes an autosomal recessive short stature syndrome characterized by alterations in the distribution of fat, intellectual disabilities, and skeletal abnormalities including a short stature and femoral neck, thickened ribs, plump long bones, and distinct facial features [56]. The homozygous mutation in XYLT1 gives rise to the substitution of the amino acid, p.Arg481Trp in the deduced catalytic domain, which results in decorin without a DS side chain in addition to mature decorin-PG with a DS chain from the fibroblasts of the patient [56]. In addition, the mutant XYLT1 is diffusely localized in the cytoplasm and partially in the Golgi in the fibroblasts of the patient.

Desbuquois dysplasia type 2 is a multiple dislocation group of skeletal disorders that is characterized by a short stature, joint laxity, and advanced carpal ossification [57]. Five distinct XYLT1 mutations have been identified to date, including a missense substitution (p.Arg598Cys), nonsense mutation (p.Arg147X), truncated form mutation (p.Pro93AlafsX69), and two splice site mutations [58]. Furthermore, fibroblasts from the affected individuals synthesized a smaller amount of CS and/or DS than those from healthy controls [58].

5.3. B4GALT7 (GalT-I)

Ehlers-Danlos syndrome is a heterogeneous group of heritable connective tissue disorders characterized by joint and skin laxity as well as tissue fragility. Six major types (classical, hypermobility, vascular, kyphoscoliosis, arthrochalasia, and dermatosparaxis types) and several minor types, including the progeroid type, are currently known [255]. Mutations in B4GALT7 encoding GalT-I cause Ehlers-Danlos syndrome-progeroid type 1, which is characterized by an aged appearance, hypermobile joints, loose yet elastic skin, hypotonic muscles, craniofacial dysmorphism, a short stature, developmental delays, generalized osteopenia, and defective wound healing [6164]. Galactosyltransferase activity is reduced in the mutant enzymes, p.Arg270Cys, p.Ala186Asp, p.Leu206Pro, and p.Arg270Cys, which results in the lack of DS side chains on decorin and biglycan core proteins and also smaller CS and HS side chains on other PGs [6468].

A homozygous mutation in B4GALT7 (p.Arg270Cys) causes a variant of Larsen syndrome in Reunion Island in the southern Indian Ocean, which is called Larsen of Reunion Island syndrome, and is characterized by distinctive facial features, multiple dislocations, dwarfism, and hyperlaxity [69].

5.4. B3GALT6 (GalT-II)

Ehlers-Danlos syndrome-progeroid type 2 is caused by mutations in B3GALT6 encoding GalT-II [70, 71]. GalT-II activity by the mutant enzyme (p.Ser309Thr) is significantly decreased, leading to the loss of GAG chains on the core proteins of various PGs [70]. The autosomal-recessive disorder, spondyloepimetaphyseal dysplasia with joint laxity type 1, which is characterized by hip dislocation, elbow contracture, clubfeet, platyspondyly, hypoplastic ilia, kyphoscoliosis, metaphyseal flaring, and craniofacial dysmorphisms such as prominent eyes, blue sclera, a long upper lip, and small mandible with cleft palate, is also caused by mutations in B3GALT6 [7072, 256]. Skeletal and connective abnormalities in both Ehlers-Danlos syndrome-progeroid type 2 and spondyloepimetaphyseal dysplasia with joint laxity type 1 overlap; however, these individuals have no common mutations among fifteen different mutations [70]. The GalT-II activities of the recombinant enzymes, p.Ser65Gly-, p.Pro67Leu-, p.Asp156Asn-, p.Arg232Cys-, and p.Cys300Ser-B3GALT6, were shown to be significantly lower than those of wild-type-B3GALT6 [70]. The mutation that affected the initiation codon, c.1A>G (p.Met1?), for B3GALT6 resulted in a lower molecular weight of the recombinant protein than that of the wild-type protein with the deletion of 41 amino acids at the N-terminus, which indicated a shift in translation at the initiation codon at the second ATG [70]. Although wild-type B3GALT6 is expressed in the Golgi, the mutant enzyme (p.Met1?) is localized in the nucleus and cytoplasm [70], indicating that the mutant protein may not be functional due to its cellular mislocalization.

5.5. B3GAT3 (GlcAT-I)

A mutation (p.Arg277Gln) in the B3GAT3 gene encoding GlcAT-I is known to cause Larsen-like syndrome [73, 74], which is characterized by dislocations in the hip, knee, and elbow joints, equinovarus foot deformities, and craniofacial dysmorphisms such as a flattened midface, depressed nasal bridge, hypertelorism, and a prominent forehead [257, 258]. These patients mainly have elbow dislocations with congenital heart defects including a bicuspid aortic valve in addition to characteristic symptoms of Larsen-like syndrome [73]. The p.Arg277Gln mutation results in a marked reduction in GlcAT-I activity in the fibroblasts of these patients and the recombinant enzyme protein [73]. Mature decorin-PG, which is secreted by fibroblasts and has a single DS side chain, was observed in the fibroblasts of healthy controls [73]. On the other hand, fibroblasts from patients generate both a PG form of decorin and DS-free decorin [73]. Moreover, the number of CS and HS in the patients’ cells is also reduced.


Neuropathies including Guillan-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hereditary motor sensory neuropathy, and unknown etiologies are partially caused by mutations in CSGALNACT1 encoding GalNAcT-I and GalNAcT-II [83]. The GalNAcT-II activities of the recombinant enzymes, CSGalNAcT1-His234Arg and -Met509Arg, were shown to be markedly reduced [83], which indicated that affect in CS chains on PGs in the nervous system may lead to peripheral neuropathies.

5.7. CHSY1

Patients with mutations in CHSY1 have Temtamy preaxial brachydactyly syndrome, which is an autosomal recessive congenital syndrome characterized by facial dysmorphism, dental anomalies, brachydactyly, hyperphalangism, growth retardation, deafness, and delayed motor and mental developments [77, 78]. Their mutations result in the substitution of amino acids and truncation of the CHSY1 protein including p.Gly5AlafsX30, p.Gly19-Leu28del, p.Glu33SerfsX1, p.Gln69X, and p.Pro539Arg and a splice-site mutation [7779]. A heterozygous mutation in CHSY1 (p.Phy362Ser) was recently identified in a patient with neuropathy [80].

5.8. CHST3 (C6ST1)

Spondyloepiphyseal dysplasia Omani type, which is characterized by severe chondrodysplasia with major involvement of the spine, is caused by a loss-of-function mutation in C6ST1 [108113]. Patients with the substitution of amino acid in C6ST1, p.Arg304Gln, have severe kyphoscoliosis, a short stature, mild brachydactyly, rhizomelia, fusion of the carpal bones, and osteoarthritis in the elbow, wrist, and knee joints [108, 109]. Furthermore, additional clinical features, including deafness, metacarpal shortening, and aortic regurgitations due to ventricular septal, mitral, and/or tricuspid defects, have been reported in Turkish siblings who had different mutations in C6ST1 (p.Tyr141Met and p.Leu286Pro) [110, 111]. Mutant enzymes of the recombinant C6ST1 and enzymes from the patients’ fibroblasts had markedly reduced C6ST activity, which resulted in the loss of chondroitin 6-O-sulfate in the fibroblasts [109111]. Moreover, chondrodysplasia with multiple dislocations, Desbuquois syndrome, autosomal recessive Larsen syndrome, and humero-spinal dysostosis have been attributed to distinct CHST3 mutations (p.Leu259Pro, p.Arg222Trp, p.Leu307Pro, p.Tyr201X, p.F206X, p.Glu372Lys, p.Gly363AlafsX30, and a mutation at the splice site) [112, 113]. Different pathological diagnoses may be caused by the relatively narrow clinical features and age-related descriptions of the same conditions [113].

5.9. CHST14 (D4ST1)

Ehlers-Danlos syndrome musculocontractural type 1, which is characterized by progressive joint and skin laxity, multiple congenital contractures, progressive multi-system complications, and characteristic craniofacial features, is caused by mutations in CHST14 encoding D4ST1 (p.Val49X, p.Lys69X, p.Pro281Leu, p.Cys289Ser, p.Tyr293Cys, and p.Glu334GlyfsX107) [97102]. A recent study described a case of Ehlers-Danlos syndrome musculocontractural type 1 (p.Val49X) in which muscle hypoplasia and weakness was observed, which resulted in myopathy based on laboratory findings such as muscle biopsy, ultrasound, and electromyography [103].

The recombinant mutants of D4ST1 (p.Pro281Leu, p.Cys289Ser, and p.Tyr293Cys) and fibroblasts from affected individuals have markedly reduced sulfotransferase activity [99]. Furthermore, a single DS side chain on decorin-PG from the fibroblasts of patients was found to be replaced by a CS chain, but not dermatan [99]. Immature decorin-PG results in the dispersion of collagen bundles in the dermal tissues of patients.

The autosomal recessive disorder, adducted thumb-clubfoot syndrome, which is characterized by an adducted thumb, clubfoot, craniofacial dysmorphism, arachnodactyly cryptorchidism, an atrial septal defect, kidney defects, cranial ventricular enlargement, and psychomotor retardation, is also caused by mutations in CHST14 (p.Val49X, p.Arg135Gly, p.Leu137Gln, p.Arg213Pro, and p.Tyr293Cys) [104106]. The fibroblasts of these patients lack DS chains and have an excess amount of CS chains.

5.10. DSE

A mutation in DSE (p.Ser268Leu) has been shown to cause Ehlers-Danlos syndrome musculocontractural type 2 [87]. Clinical features including hypermobility of the finger, elbow, and knee joints, characteristic facial features, contracture of the thumbs and feet, and myopathy have been observed in these patients. Epimerase activity is markedly reduced not only in the recombinant mutant DSE (p.Ser268Leu) but also in the cell lysate from these patients [87]. In addition, a decrease in the biosynthesis of DS accompanied by an increase in that of CS has been reported in the fibroblasts of these patients. The deficiencies associated with DSE in addition to D4ST1 affect the biosynthesis of DS, which implies that both enzymes are essential for the development of skin and bone as well as the maintenance of their extracellular matrices.

6. Conclusions

The biological roles of CS, DS, and HS chains in vivo have been revealed by examining knockout mice in addition to nematodes, fruit flies, and zebrafish [4, 8, 1214]. However, the mice deficient in glycosyltransferases or sulfotransferases involved in the biosynthesis of GAGs showed embryonic lethality or death shortly after the birth. These observations indicate that GAGs or PGs are essential for early development. Furthermore, studies using the conditional knockout mice have revealed the specific functions of GAGs in individual organs. Recent advances in the study of human genetic diseases in the bone and connective tissue have also clarified the biological significance of the GAG side chains of PGs [7, 14, 20]. The clinical manifestations in human disorders caused by deficiency in the biosynthetic enzymes of GAGs do not always agree with the phenotypes of the deficiency in the corresponding enzymes in knockout mice. This contradiction may be due to the residual enzymatic activity or GAGs in human patients. Although null mutant mice show severe phenotypes including embryonic lethality, human patients appear to show various symptoms depending on the degree of remaining activity of the enzymes. Further comprehensive approaches to the study of molecular pathogeneses involving CS, DS, and HS chains are required to facilitate the development of therapeutics and design of new drugs for these diseases.


B3GALT6:Beta 1,3-galactosyltransferase 6
B4GALT7:Beta 1,4-galactosyltransferase 7
B3GAT3:Beta-1,3-glucuronyltransferase 3
C4ST:Chondroitin 4-O-sulfotransferase
C6ST:Chondroitin 6-O-sulfotransferase
CHST:Carbohydrate sulfotransferase
CHSY:Chondroitin synthase
CS:Chondroitin sulfate
CSGALNACT:Chondroitin sulfate N-acetylgalactosaminyltransferase
D4ST:Dermatan 4-O-sulfotransferase
DS:Dermatan sulfate
DSE:Dermatan sulfate-glucuronyl C5-epimerase
DSEL:Dermatan sulfate epimerase-like
GalNAc: N-Acetyl-D-galactosamine
GalNAc4S-6ST: N-Acetyl-D-galactosamine 4-sulfate 6-O-sulfotransferase
GlcUA: D-Glucuronic acid
HS:Heparan sulfate
IdoUA: L-Iduronic acid
PAPS:3′-Phosphoadenosine 5′-phosphosulfate
PAPSS2:3′-Phosphoadenosine 5′-phosphosulfate synthase 2

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas 24110501 (to Kazuyuki Sugahara) and 26110719 (to Shuhei Yamada) from The Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); by a Grant-in-Aid for Scientific Research (C) 24590071 (to Shuhei Yamada); by a Grant-in-Aid for Young Scientists (B) 25860037 (to Shuji Mizumoto) from the Japan Society for the Promotion of Science, Japan; by the Drs. Hiroshi Irisawa and Aya Irisawa Memorial Research Grant from the Japan Heart Foundation (to Shuji Mizumoto); by the Research Institute of Meijo University (Tenkai) (to Shuji Mizumoto); and by the Fugaku Trust for Medical Research (to Shuhei Yamada).