Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs.

A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs.
Proteoglycans (PGs) 2 having linear polysaccharides as side chains are widely distributed in extracellular matrices and at cell surfaces (1)(2)(3). Chondroitin sulfate (CS) and dermatan sulfate (DS) chains are classified as sulfated glycosaminoglycans (GAGs) and are covalently attached to the core proteins of PGs (1)(2)(3). PGs function in embryonic development and play roles in the pathological development of a number of diseases through the GAG chains (3)(4)(5)(6)(7). GAGs are modified by sulfation at various positions of hydroxy groups in each constituent sugar residue and by epimerization of uronic acid residues during the biosynthetic process, resulting in enormous structural diversity, which is fundamental to a wide range of biological events involving GAGs (4). Thus, it is imaginable that the heritable disturbance of the fine structure of GAGs may cause a variety of diseases.
The backbones of CS and DS consist of repeating disaccharide building units of GalNAc and uronic acid, D-glucuronic acid (GlcUA), or L-iduronic acid (IdoUA). CS/DS hybrid chains with both CS and DS structural elements are often found in mammalian tissues and are modified by sulfate groups at C-2 of uronic acids and at C-4 and/or C-6 of GalNAc residues with various combinations (4). In recent years, most (if not all) glycosyltransferases/epimerases/sulfotransferases and related enzymes for GAG biosynthesis have been cloned and characterized (Figs. 1 and 2 and Table 1) (3,7,8), although their regulatory mechanism(s) at the transcriptional level are largely not yet understood. However, in addition to well established mucopolysaccharidoses and lysosomal storage diseases (9), which are characterized by the accumulation of undigested GAG fragments in lysosomes due to defective catabolism by mutated glycosidases and sulfatases, several genetic diseases caused by mutations of the genes encoding biosynthetic enzymes have recently been described. Examples include hereditary multiple exostoses resulting from mutations in the EXT1 and EXT2 genes, encoding the glycosyltransferases responsible for heparan sulfate (HS) biosynthesis (10,11); chondrodysplasias caused by mutations in the sulfate transporter and 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) synthase-2 (12); and the Ehlers-Danlos syndrome (EDS) progeroid form caused by mutations in B4GALT7, encoding ␤4-galactosyltransferase-7, resulting in a defect in DS chains (13)(14)(15)(16)(17)(18). Accumulating evidence suggests that, in addition to the abovementioned genes, CS/DS biosynthetic enzymes are crucial to bone development and skin integrity in humans ( Table 2). This minireview will overview the biosynthetic mechanism for CS/DS chains and focus on genetic diseases that have been recently characterized from a glycobiological point of view in terms of disturbances in the biosynthesis of functional CS/DS chains.

Biosynthesis of CS and DS Chains
GAG-Protein Linkage Region-The newly synthesized core proteins of PGs are initially modified by glycosylation to form a common GAG-protein linkage region tetrasaccharide, GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-(GlcUA-Gal-Gal-Xyl-), attached to the serine residue(s) of the GAG attachment sites of the PGs in the endoplasmic reticulum and Golgi compartments (2,4,7). Each specific glycosyltransferase, ␤-xylosyltransferase (XylT) (19,20), ␤1,4-galactosyltransferase-I (GalT-I) (21,22), ␤1,3-galactosyltransferase-II (GalT-II) (23), and ␤1,3-glucuronosyltransferase (GlcAT)-I (24), which are encoded by XYLT1 (and XYLT2), B4GALT7, B3GALT6, and B3GAT3, respectively, transfers to the serine residue or growing glycan FIGURE 1. Schematic presentation of the biosynthetic assembly of the GAG backbones by various glycosyltransferases. Each glycosyltransferase requires the respective UDP-sugar as a donor substrate. Following the synthesis of specific core proteins, the synthesis of the so-called GAG-protein linkage region, GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-, common to CS/DS and HS/heparin (Hep) chains, is initiated by XylT, which transfers a Xyl residue from UDP-Xyl to the specific Ser residue in the endoplasmic reticulum, and is completed by the consecutive addition of each sugar by GalT-I, GalT-II, and GlcAT-I, which are common to the biosynthesis of both CS and HS, in the Golgi apparatus. Following completion of the synthesis of the linkage region, the first ␤GalNAc residue is transferred to the naked GlcUA residue in the linkage region by GalNAcT-I, which initiates the assembly of the chondroitin backbone. Subsequently, the repeating disaccharide region, (-3GalNAc␤1-4GlcUA␤1-) n , is elongated by alternate additions of GlcUA and GalNAc residues from UDP-GlcUA and UDP-GalNAc catalyzed by CS-GlcAT-II and GalNAcT-II activities, respectively, of a heterocomplex (CS polymerase) formed by ChSy and ChPF. On the other hand, the addition of ␣1-4-linked GlcNAc to the linkage region by GlcNAcT-I initiates the assembly of the HS repeating disaccharide region, (-4GlcNAc␣1-4GlcUA␤1-) n . Then, the chain polymerization of the HS chain is catalyzed by HS-GlcAT-II and GlcNAcT-II activities of HS polymerase, which is a heterocomplex of EXT1 and EXT2. The molecular mechanism of the differential biosynthetic assembly of HS and CS chains at the GAG attachment sites remains to be elucidated, as details have been discussed in the text; and therefore, the transfer reactions of the fifth sugar (first amino sugar) are shown in this figure by the dashed and dashed-dotted arrows. After the formation of the chondroitin and heparan backbones, GAG chains are matured by sulfation at various positions and epimerization at GlcUA residues. Each enzyme (glycosyltransferase and/or epimerase), its coding gene, and the corresponding inherited disorder are described under the respective sugar symbols. Sulfotransferases involved in the chain modifications are not included but are illustrated in Fig. 2 (see also Table 2 for the inherited diseases of sulfotransferases). DSE, dermatan sulfate epimerase; DSEL, dermatan sulfate epimerase-like.

Linkage region
XylT

Repeating disaccharide region
Chondroitin synthase (GalNAcT-II, and CS-GlcAT-II) from the corresponding UDP-sugars, including UDP-Xyl, UDP-Gal, and UDP-GlcUA (Table 1). The GAG-protein linkage region tetrasaccharides (GlcUA-Gal-Gal-Xyl-O-) of CS and HS might be synthesized by the same set of enzymes, including XylT, GalTs, and GlcAT-I, some of which may form a multienzyme complex such as a so-called GAGosome for HS synthesis (25,26). Furthermore, the sugar residues in the GAG linkage region are frequently modified by 2-O-phosphorylation (the xylose residue) and sulfation at C-6 (the first galactose) and C-4 or C-6 (the second galactose) (2). The enzymes responsible for the phosphorylation and sulfation have been identified as FAM20B and chondroitin 6-O-sulfotransferase-1 (C6ST-1), respectively (27,28). Although the biological functions of these modifications remain unclear, they influence, at least in vitro, the glycosyltransferase activities of GalT-I and GlcAT-I. Repeating Disaccharide Region of CS/DS-Following completion of the building of the tetrasaccharide GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-Ser, the first GalNAc residue is transferred to the GlcUA residue in the linkage region by ␤1,4-N-acetylgalactosaminyltransferase (GalNAcT)-I, resulting in initiation of the synthesis of the repeating disaccharide region of CS/DS chains ( Fig. 1 and Table 1) (29 -32). Alternatively, the addition of a GlcNAc residue to the linkage region by ␣1,4-Nacetylglucosaminyltransferase (GlcNAcT)-I evokes HS biosyn-  (Fig. 1) (33-41). Thus, the transfer of the first hexosamine residue, ␣GlcNAc, or ␤GalNAc, which is the fifth saccharide from the reducing terminal, is crucial in determining the type of GAGs as HS or CS. The biosynthesis of a HS chain on core proteins requires a cluster of acidic and hydrophobic amino acids located near Ser-Gly of the GAG attachment site (41). In addition, the sulfation of Gal residues in the GAG-protein linkage region (GlcUA-Gal-Gal-Xyl-O-) has been reported (2). The potential sites for sulfation are C-6 of the first Gal residue and C-4 or C-6 of the second Gal residue, which occurs in the linkage region of CS/DS, but not HS/heparin (2). These observations indicate that the amino acids of the core protein at the GAG attachment site, the sulfation of the linkage region, and/or unknown additional factors may be involved in the selective assembly of CS and HS chains. Thus, the molecular mechanism of the enigmatic differential biosynthetic assembly of HS and CS chains at the GAG attachment sites remains a black box.
Thereafter, polymerization of the CS backbone occurs to construct the repeating disaccharide region consisting of -3GalNAc␤1-4GlcUA␤1-by enzymatic activities designated as CS-GlcAT-II and GalNAcT-II and catalyzed by a CS polymerase enzyme complex composed of various combinations of the chondroitin synthase family, including chondroitin synthase (ChSy), chondroitin polymerizing factor (ChPF), and the other four family members (Table 1) (42)(43)(44)(45)(46)(47)(48). ChSy consists of 802 amino acids with homology to ␤3-galactosyltransferase and ␤4-galactosyltransferase family members on the N-and C-terminal sides, respectively, and is a bifunctional glycosyltransferase with GalNAcT-II and CS-GlcAT-II activities required for the formation of the disaccharide unit (42). On the other hand, ChPF possesses only weak GalNAcT-II activity (43), but Yada et al. (47) independently reported that ChPF has both GalNAcT-II and CS-GlcAT-II activities, resulting in the designation of ChPF as chondroitin sulfate synthase-2 (CSS2). Despite the dual enzymatic activities of ChSy, ChSy itself cannot achieve polymerization reactions to build up the repeating disaccharide units of CS. However, the association of ChSy with ChPF results in a dramatic augmentation of both glycosyltransferase activities of ChSy (43). Furthermore, this enzyme complex can polymerize a CS chain onto the linkage region tetrasaccharide attached to the core protein (43). Thus, ChPF may function as a chaperone, which confers on ChSy the stronger glycosyltransferase activities or stabilizes ChSy by forming a ChSy-ChPF enzyme complex (43)(44)(45).
The characteristics of the progeroid type of EDS (GalT-I deficiency) include an aged appearance, developmental delay, short stature, craniofacial dysmorphism, generalized osteopenia, defective wound healing, hypermobile joints, hypotonic muscles, and loose yet elastic skin (13)(14)(15)(16). Fibroblasts from these patients with the R270C mutation in GalT-I show reduced galactosyltransferase activity compared with control subjects and synthesize deglycanated decorin and biglycan core proteins in addition to their PG forms (16). It has also been demonstrated that the A186D mutation markedly reduces GalT-I activity in vitro, whereas its effects on the biosynthesis of CS/DS and HS are much less pronounced (17). In addition, a drastic decrease in GalT-I activity and GAG biosynthesis caused by L206P and R270C mutations has been reported (63,64). Interestingly, the reduction in GalT-I activity caused by the R270C mutation results in a reduction in the sulfation of HS chains and a retardation of wound closure in vitro (18). Taken together, the phenotypes of the EDS progeroid form caused by GalT-I mutations are attributable to defects in mainly DS and partially HS and/or CS chains.
GlcAT-I (B3GAT3) Deficiency-A family with recessive inheritance and five affected individuals with joint dislocations affecting mainly the elbow and congenital heart defects, including a bicuspid aortic valve, was reported. A mutation (R277Q) in the B3GAT3 gene coding for GlcAT-I was identified for this Larsen-like syndrome family (65). Larsen syndrome is characterized by dislocations of the hip, knee, and elbow joints; equinovarus foot deformity; and craniofacial dysmorphism that includes hypertelorism, prominence of the forehead, a depressed nasal bridge, and a flattened midface (66,67). The R277Q mutation causes a drastic reduction in GlcAT-I activity in the patients' fibroblasts (ϳ5% of control fibroblasts) (65). Although wild-type GlcAT-I is located in the cis and cis-medial Golgi in control fibroblasts, the amount of mutant protein is markedly reduced as demonstrated by immunofluorescent staining using anti-GlcAT-I antibody, indicating that the GlcAT-I mutant may be produced to a lesser extent, be degraded, or be susceptible to a protease compared with the wild type (65). Furthermore, the mutation results in a decrease in the biosynthesis of GAGs. Fibroblasts from patients produce not only a PG form of decorin, which is secreted by the fibroblasts and has a single DS chain, but also DS-free decorin presumably bearing the linkage region trisaccharide stub Gal␤1-3Gal␤1-4Xyl (65). Furthermore, the numbers of CS and HS chains on the core proteins at the surface of the fibroblasts are reduced to 65 and 53% of those in control subjects, respectively (65). These observations suggest that the GlcAT-I mutant (R277Q) cannot transfer GlcUA to the common GAG-protein linkage region trisaccharide Gal-Gal-Xyl, resulting in a partial deficiency in CS, DS, and HS that presents as connective tissue disorders with heart defects and Larsen-like syndrome (B3GAT3-type).
More recently, another mutation (P140L) was found in a consanguineous family from the Nias island in Indonesia (68). These patients had skeletal phenotypes characterized by disproportionate short stature but no heart phenotype in contrast to the R277Q mutation. A recombinant enzyme of the P140L mutation showed significant reduction in enzymatic activity, reflecting the mutation that lies within the donor substratebinding subdomain of the catalytic domain of GlcAT-I. However, cultured lymphoblastoid cells show that defective synthesis is more pronounced for CS than for HS.
CSGALNACT1 Deficiency-Two possible mutations in the CSGALNACT1 gene, encoding a protein with GalNAcT-I and GalNAcT-II activities, are found in patients with Bell palsy and an unknown type of hereditary motor and sensory neuropathy (69). Hereditary motor and sensory neuropathies are heterogeneous neurodegenerative disorders characterized by a progressive loss of function in the peripheral sensory nerves (70). Symptoms commonly include weakness, falls, and sensory loss often associated with cavus or planus foot deformity (70). Degeneration of myelin sheaths and/or axons causes paralytic amyotrophy predominantly involving distal limbs in association with hypo-or areflexia. The recombinant mutant proteins for H234R and M509R exhibit no GalNAcT-II activity, implying that these mutations in CSGALNACT1 and/or CS PGs may be associated with pathogenetic mechanisms of the peripheral neuropathies (69). To further understand the neuropathy involving CS, knock-out mice (Csgalnact1 Ϫ/Ϫ ) may be useful, although currently, the mice are reported to show only abnormal development in cartilage (71,72).
CHSY1 Deficiency-The Temtamy pre-axial brachydactyly syndrome is an autosomal recessive congenital syndrome characterized by bilateral symmetric pre-axial brachydactyly and hyperphalangism, facial dysmorphism, dental anomalies, sensorineural hearing loss, delayed motor and mental developments, and growth retardation. The disease is caused by mutations in CHSY1 (chondroitin synthase-1), including Gly-19 -Leu-28del, G5Afs*30, Gln-69*, and P539R (73,74). The knockdown of chsy1 in zebrafish suggests that it is involved in the signaling of bone morphogenetic protein during bone development (73). Tian et al. (74) reported syndromic recessive pre-axial brachydactyly with partial duplication of proximal phalanges caused by CHSY1 mutations. Furthermore, Wilson et al. (75) recently demonstrated that Chsy1 knock-out (Chsy1 Ϫ/Ϫ ) mice manifest brachypodism, with a striking patterning defect in distal phalanges, chondrodysplasia, and a decrease in bone density. Associated with the digit-patterning defect are a reduction in CS and a shift in cell orientation. The expression of Gdf5 (growth and differentiation factor 5), a member of the bone morphogenetic protein family, is altered during the earliest stages of joint formation in the Chsy1 Ϫ/Ϫ mouse (75), indicating that Chsy1 restricts Gdf5 expression. These observations suggest that CHSY1 and/or CS chains are indispensable regulators of joint patterning and skeletal development and that the Chsy1 Ϫ/Ϫ mouse is a good animal model for human brachydactyly caused by CHSY1 mutations.
C6ST-1 (CHST3) Deficiency-A loss-of-function mutation in C6ST-1 causes human Omani-type spondyloepiphyseal dysplasia, a severe chondrodysplasia with major involvement of the spine (76 -81). The original patients with Omani-type spondyloepiphyseal dysplasia caused by a missense mutation (R304Q) had a short stature; severe kyphoscoliosis; osteoarthritis in elbow, wrist, and knee joints; secondary dislocation of the large joints; rhizomelia; fusion of carpal bones; and mild brachydactyly (76,77). Several of their clinical features (including ventricular septal, mitral, and/or tricuspid defects; aortic regurgitations; deafness; and metacarpal shortening) differed significantly from the original description of the disease in Turkish siblings (T141M and L286P) (78,79). 6-O-Sulfation on GalNAc residues in CS chains was barely detected in fibroblasts and urine obtained from the patients (78). Furthermore, Superti-Furga and colleagues (80,81) have demonstrated that additional CHST3 mutations cause autosomal recessive Larsen syndrome, chondrodysplasia with multiple dislocations, humerospinal dysostosis, and Desbuquois syndrome. These observations suggest that the degree of 6-O-sulfation deficiency in CS varies depending on the substituted amino acids in C6ST-1. The clinical spectra are similar to those seen in other skeletal dysplasias caused by defective sulfation of GAGs. Different pathological phenotypes may result from relatively narrow clinical features and age-related descriptions of the same conditions.
D4ST-1 (CHST14) Deficiency-Kosho et al. (82,83) reported six unrelated Japanese patients showing characteristic craniofacial features, multiple congenital contractures, progressive joint and skin laxity, and progressive multisystem complications, features partially similar to those of kyphoscoliosis type VI EDS, caused by a deficiency in lysyl hydroxylase. Although lysyl hydroxylase activity was normal in these patients, homozygosity mapping of two independent consanguineous families identified CHST14 encoding D4ST-1 harboring four mutations (Lys-69*, P281L, C289S, and Y293C) (84). Recombinant mutant D4ST-1 showed no D4ST activity (84). In addition, the fibroblasts from the patients showed a marked reduction in sulfotransferase activity (84). Surprisingly, CS chains (but not dermatan) were produced as decorin side chains by the fibroblasts (84). In fact, 4-O-sulfations in CS and DS chains act as a block to prevent DS epimerase from re-equilibrating between GlcUA and IdoUA (50). Hence, the defect in D4ST-1 allows a back-epimerization reaction converting IdoUA to GlcUA to form chondroitin, followed by sulfation with C4ST, resulting in an aberrant shift from DS to CS synthesis, which may affect the formation or maintenance of adequate collagen bundles in patient dermal tissues (84).
Dündar et al. (85) and Malfait et al. (86) independently reported that the mutations in D4ST-1 caused adducted thumb-clubfoot syndrome (ATCS) and musculocontractural type EDS (EDS type VIB) without a mutation in lysyl hydroxylase. ATCS is an autosomal recessive disorder showing characteristic clinical features such as adducted thumb, clubfoot, craniofacial dysmorphism, arachnodactyly cryptorchidism, atrial septal defect, kidney defect, cranial ventricular enlargement, and psychomotor retardation, as well as thin and translucent skin, joint instability, and osteopenia from birth to early childhood (87,88). Five of the 11 patients with ATCS died in early infancy or childhood, indicating that ATCS patients may have more severe manifestations than patients with EDS type VIB.

Conclusion
The cloning of cDNAs for the genes encoding enzymes involved in the biosynthesis of GAG chains during the last 15 years has led to a better understanding of not only the biosynthetic mechanism but also the functions of CS, DS, and HS chains in vivo, which have been clarified by using model organisms such as nematodes, fruit flies, zebrafish, and knock-out mice (7, 8, 89 -91). Moreover, recent advances in the study of human genetic diseases of the skeleton and skin achieved by the cooperative efforts of clinicians, molecular geneticists, and glycobiologists have revealed the importance of CS/DS side chains of PGs. A further understanding of the molecular pathogenesis involving CS and DS chains is essential to facilitate the development of therapeutics for these diseases.