Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

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.


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 [1][2][3][4][5][6][7]. 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 [7][8][9][10][11][12][13][14][15][16][17][18].
Glycosyltransferases, epimerases, sulfotransferases, and related enzymes in the biosynthesis of GAGs have been cloned and characterized (Tables 1-4 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. HS and heparin consist of uronic acid and GlcNAc residues with varying proportions of IdoUA. Heparin is highly sulfated and has a large proportion of IdoUA residues, whereas HS is low sulfated and has a high proportion of GlcUA. These sugar moieties are esterified by sulfate at various positions as indicated by the circled "S. " The abbreviation of "i" in iO, iA, iB, iC, iD, and iE stands for IdoUA. HexUA represents hexuronic acid (GlcUA or IdoUA).

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 [21][22][23]. 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].  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 (            Figure 2: Biosynthetic assembly of GAG backbones by various glycosyltransferases. All glycosyltransferases require a corresponding UDPsugar, such as UDP-Xyl, -Gal, -GlcUA, -GalNAc, and -GlcNAc, as a donor substrate. After specific core proteins have been synthesized, the synthesis of the common GAG-protein linkage region, GlcUA 1-3Gal 1-3Gal 1-4Xyl 1-, is evoked by XylT, which transfers a Xyl residue from UDP-Xyl to the specific serine (Ser) residue(s) at the GAG attachment sites. The linkage tetrasaccharide is subsequently constructed by GalT-I, GalT-II, and GlcAT-I. These four enzymes are common to the biosynthesis of CS, DS, HS, and heparin. The first 1-4-linked GalNAc residue is then transferred to the GlcUA residue in the linkage region by GalNAcT-I, which initiates the assembly of the chondroitin backbone, thereby resulting in the formation of the repeating disaccharide region, [-3GalNAc 1-4GlcUA 1-] n , by CS-polymerase. Alternatively, the addition of 1-4-linked GlcNAc to the linkage region by GlcNAcT-I initiates the assembly of the repeating disaccharide region [-4GlcNAc 1-4GlcUA 1-] n of HS and heparin by HS-polymerase. Following the formation of the chondroitin and heparan backbones, both precursor chains are modified by sulfation and epimerization (see Figure 3). Each enzyme, its coding gene, and the corresponding inheritable disorder are described under the respective sugar symbols from the top of each line. SEMDJL1, spondyloepimetaphyseal dysplasia with joint laxity type 1. 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 1,4-N-acetylgalactosaminyltransferase-I (GalNAcT-I) ( Figure 2) [193][194][195][196]. Alternatively, the transfer of a GlcNAc residue from UDP-GlcNAc to the linkage region tetrasaccharide by 1,4-N-acetylglucosaminyltransferase-I (GlcNAcT-I) is known to result in the initiation of the repeating disaccharide region of HS and heparin chains ( Figure 2) [197][198][199][200][201]. Six chondroitin synthase family members have been identified including chondroitin synthases (ChSys), chondroitinpolymerizing factor (ChPF), and CSGalNAcTs ( Figure 2 and Table 3) [193][194][195][196][202][203][204][205][206][207][208]. 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, -4GlcUA 1-3GalNAc 1 ( 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 [203][204][205]. 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) [210][211][212], 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].

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]. 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 1 −/− mice led to better recovery from injuries in the nervous system than the wild type.  to be markedly reduced in the spleens and brains of C6st1deficient 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.

B3gat3 (GlcAT-I). Mice deficient in
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. 4.9. 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 Dunit (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. (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.

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 [119][120][121]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 [124][125][126]. 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 [128][129][130][131][132]. 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 tissuespecific manner.

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 CCl 4 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 isletcells 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.

Ndst1, 2, and 3. Functional analyses of HS and heparin
using Ndst1-deficient mice have been performed in approximately 20 studies to date . 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 [140-142, 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 [165][166][167]. These findings indicated that the storage of proteases in granules is controlled by heparin or heparin-PG, such as serglycin [165][166][167]. On the other hand, Ndst3-deficient mice develop normally and are fertile [168].

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 CXCL12 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 [173][174][175][176][177][178][179]. 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].

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].

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].

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-Osulfation by Sulfs may control multiple functions of HS chains during morphogenesis. 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.

Human Disorders Affecting the Skeleton and Skin due to the Disturbance of GAGs
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 [46][47][48]. 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].

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].
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].

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 [70][71][72]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.

B3GAT3 (GlcAT-I).
A mutation (p.Arg277Gln) in the B3GAT3 gene encoding GlcAT-I is known to cause Larsenlike 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. 5.6. CSGALNACT1. 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.
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.

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.

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,[12][13][14]. 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. l-Iduronic acid PAPS: 3 -Phosphoadenosine 5 -phosphosulfate PAPSS2: 3 -Phosphoadenosine 5 -phosphosulfate synthase 2 PG: Proteoglycan.

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