Loss of dermatan sulfate epimerase (DSE) function results in ...

Loss of dermatan sulfate epimerase (DSE) functionresults in musculocontractural Ehlers–Danlossyndrome

Thomas Muller1,{, Shuji Mizumoto4,{, Indrajit Suresh5,{, Yoshie Komatsu4, Julia Vodopiutz6,

Munis Dundar7, Volker Straub8, Arno Lingenhel2, Andreas Melmer3, Silvia Lechner2,

Johannes Zschocke2, Kazuyuki Sugahara4 and Andreas R. Janecke1,2,∗

1Department of Pediatrics I, 2Division of Human Genetics and 3Department of Internal Medicine I, Innsbruck Medical

University, Innsbruck, Austria, 4Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for

Post- Genomic Science and Technology, Graduate School of Life Science, Hokkaido University, Sapporo, Japan,5Jagadguru Sri Shivarathreeshwara University Hospital, Mysore, Karnataka, India, 6Department of Pediatrics and

Adolescent Medicine, Medical University of Vienna, Vienna, Austria, 7Department of Medical Genetics, Erciyes

University, Talas, Kayseri, Turkey and 8Institute of Genetic Medicine, Newcastle University, Central Parkway,

Newcastle upon Tyne NE1 3BZ, UK

Received March 17, 2013; Revised and Accepted May 16, 2013

The sulfated polysaccharide dermatan sulfate (DS) forms proteoglycans with a number of distinct core proteins.Iduronic acid-containing domains in DS have a key role in mediating the functions of DS proteoglycans. Twotissue-specific DS epimerases, encoded by DSE and DSEL, and a GalNAc-4-O-sulfotransferase encoded byCHST14 are necessary for the formation of these domains. CHST14 mutations were previously identified forpatients with the musculocontractural type of Ehlers–Danlos syndrome (MCEDS). We now identified a homozy-gous DSE missense mutation (c.803C>T, p.S268L) by the positional candidate approach in a male child withMCEDS, who was born to consanguineous parents. Heterologous expression of mutant full-length and solublerecombinant DSE proteins showed a loss of activity towards partially desulfated DS. Patient-derived fibroblastsalso showed a significant reduction in epimerase activity. The amount of DS disaccharides was markedlydecreased in the conditioned medium and the cell fraction from cultured fibroblasts of the patient when com-pared with a healthy control subject, whereas no apparent difference was observed in the chondroitin sulfate(CS) chains from the conditioned media. However, the total amount of CS disaccharides in the cell fractionfrom the patient was increased ∼1.5-fold, indicating an increased synthesis or a reduced conversion of CSchains in the cell fraction. Stable transfection of patient fibroblasts with a DSE expression vector increasedthe amount of secreted DS disaccharides. DSE deficiency represents a specific defect of DS biosynthesis. Wedemonstrate locus heterogeneity in MCEDS and provide evidence for the importance of DS in human develop-ment and extracellular matrix maintenance.

INTRODUCTION

Proteoglycans are formed by covalent attachments of glycosa-minoglycans (GAGs), long chains composed of repeating disac-charide subunits such as dermatan sulfate (DS), chondroitin

sulfate (CS) and heparan sulfate (HS), to serine residues ofcore proteins. Proteoglycans are major components of the extra-cellular matrix and contribute to normal embryonic and post-natal development and tissue homoeostasis by ensuring tissuestability and signaling functions. For example, the negative

†The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.

∗To whom correspondence should be addressed at: Department of Pediatrics I, Division of Human Genetics, Innsbruck Medical University, Anichstrasse35, A-6020 Innsbruck, Austria. Tel: +43 51250482415; Fax: +43 51250424934; Email: [email protected]

# The Author 2013. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2013, Vol. 22, No. 18 3761–3772doi:10.1093/hmg/ddt227Advance Access published on May 23, 2013

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charge of GAGs provides hydrophilic properties, and the accu-mulating water mechanically ensures an appropriate elastictissue tonus; the small DS proteoglycans biglycan and decorinare known for their roles in forming and modifying collagenfibers in size and organization. Mature proteoglycans andunbound GAGs have also been implicated in modulatinggrowth factor signaling (1–6). DS GAGs and DS proteoglycansinteract with and modulate the activity of signaling moleculesparticipating in cell adhesion, migration, proliferation, neuriteoutgrowth, wound repair and anticoagulant processes (5,7–10). A widespread distribution of DS proteoglycans in mamma-lian tissues has been described including blood vessel walls,skin, tendon, sclera, cartilage and undifferentiated mesenchymaltissues (11). Recent studies have also revealed roles of CS/DSchains in the orchestration of the neural stem/progenitor cellmicromilieu (12).

A basic outline of CS/DS biosynthesis is summarized inFigure 1. A covalent tetrasaccharide linkage sequence (glucur-onic acid–galactose–galactose–xylose-O–) is common to theformation of HS, CS and DS proteoglycans and results fromthe actions of four distinct monosaccharide transferases (13).The type of GAGs synthesized is then defined by the compositionof the GAG chains added to the linker tetrasaccharide of the cor-responding proteoglycans (Fig. 1).

CS is synthesized by the action of CS synthases, which alter-nately add N-acetyl-D-galactosamine (GalNAc) and D-glucuronicacid (GlcUA) residues to the tetrasaccharide linkage region. DSand CS/DS hybrid GAG chains are produced by the epimerization

of the CS C-5 hydroxy of a number of GlcUA residues toL-iduronic acid (IdoUA) by glucuronyl C5-epimerases (14).Two genes encoding chondroitin-glucuronate C5-epimerases(EC 5.1.3.19), DSE (dermatan sulfate epimerase, DS-epi1,SART2, squamous cell carcinoma antigen recognized by T cell2, [MIM 605942]) and DSEL (dermatan sulfate epimerase like,DS-epi2, [MIM 611125]), respectively, are present in organismsranging from Xenopus tropicalis to humans but not in worms orflies (15). These enzymes can catalyze the epimerization of theC-5 hydroxy in both directions; the addition of sulfate to the C-4hydroxy of the adjacent GalNAc to IdoUA or GlcUA hindersfurther epimerization of these residues (16). Four GalNAc-4-O-sulfotransferases have been cloned that act on CS and/orDS, chondroitin 4-O-sulfotransferases 1–3 (CHST11 [MIM610128], CHST12 [MIM 610129], CHST13 [MIM 610124])(17–19) and dermatan-4-O-sulfotransferase 1 (CHST14 [MIM608429]) (20,21). DS and CS can be further modified by the add-ition of sulfate to the C-2 hydroxy of IdoUA and GlcUA, and toC-6 hydroxy of GalNAc (22). Both the degree of DS sulfationand the proportions of IdoUA and GlcUA vary spatiotemporarily,and increase the structural and functional diversity of proteogly-cans (10,23).

The Ehlers–Danlos syndromes (EDSs) represent a clinicallyand genetically heterogeneous group of diseases, which are char-acterized by fragility of the soft connective tissues. The clinicalspectrum varies from mild skin and joint hyperlaxity to severephysicaldisability, and to life-threateningvascularcomplications.Mutations in genes encoding fibrillar collagens or enzymes

Figure 1. Biosynthesis of DS and chondroitin sulfate (CS). CS, DS and also HS share the synthesis of a tetrasaccharide linker region that attaches the glycosamino-glycan chains to a serine residue within the conserved attachment site of core proteins. The activity of a unique N-acetylgalactosaminyltransferase-I (GalNAcT-I) thattransfers the first GalNAc residue onto the tetrasaccharide linker starts a growing glycosaminoglycan chain to CS. This step is followed by the activities of specificenzymes that polymerize the glycosaminoglycan chain by the alternate additions of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcUA) moieties inCS. CS chains can be modified during elongation by Golgi residents, epimerase and a number of sulfotransferases. After the formation of the chondroitin backbone,epimerization of GlcUAto L-iduronicacid (IdoUA)by C5-hydroxyl epimerases (DSE or DSEL) followed by sulfate addition to the C4 hydroxyof the adjacent GalNAcresidue by D4ST1 generates DS from CS, and prevents back-epimerization of IdoUA to GlcUA. In CS sequences, the C4 hydroxy of the GalNAc residue adjacent toGlcUA can be sulfated by specific CS/DS sulfotransferases C4ST1, -2 and -3. The extent of modification with sulfate groups by the actions of CS/DS sulfotransferasesvaries between different core proteins, and spatio-temporally. All sulfotransferases transfer a sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS),a universal sulfate donor to the respective sugar residue.

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involved in post-translational modification of collagens accountfor most forms of EDSs. Six EDS subtypes are currently distin-guished based on combinations of clinical symptoms, inheritancepattern and the nature of the underlying biochemical and molecu-lar defect(s) (Villefranche classification) (24). However, therecent identification of new EDS genes would justify a revisionof the Villefranche classification (25).

We recently described loss-of-function mutations in CHST14,resulting in dermatan 4-O-sulfotransferase 1 (D4ST1) deficiency,as the first disorder resulting from impaired DS biosynthesis(26,27). D4ST1 deficiency causes a clinically recognizable formof EDS, referred to as D4ST1-deficient EDS or musculocontrac-tural EDS (MCEDS; [MIM 601776]) (28). However, a numberof different terms have been used in the past for this condition.Adducted thumb-clubfoot syndrome (ATCS) (26,29–31),Kosho Type of EDS (27,32,33), Dundar syndrome, as well askyphoscoliosis EDS without lysyl hydroxylase deficiency (EDSVIB) and distal arthrogryposis with peculiar facies and hydrone-phrosis are terms that have been used to describe this disorder.The recent reporting of patients with CHST14 mutations andin-depth reviews of clinical findings support the notion that thedisorders listed above all constitute a single, clinically recogniz-able form of EDS (28,34–37). The disorder is characterized byprogressive multisystem fragility-related manifestations resultingfrom impaired assembly of collagen fibrils—i.e. joint dislocationsand deformities, skin hyperextensibility, bruisability and fragil-ity—andrecurrent large subcutaneoushematomas,and frequentlyby cardiac valvular, respiratory, gastrointestinal and ophthalmo-logical complications. Variousmalformations, i.e. distinct cranio-facial features, multiple congenital contractures, and congenitaldefects in cardiovascular, gastrointestinal, renal, ocular andcentral nervous systems, might either result from impaired assem-bly of collagen fibrils or might result from deranged signalingpathways during development. In addition, a myopathy featuringmuscle hypoplasia, muscle weakness and an abnormal musclefiber pattern on histology in adulthood has been recognized inMCEDS (38), similarly as in other EDS types (39–41), and prob-ably contributes to the gross motor developmental delay in thistype of EDS.

In this study, we identified a homozygous DSE mutationp.S268L in a patient with MCEDS. We demonstrate that the mu-tation affects the epimerase’s activity in that (i) patient-derivedfibroblasts show a significant reduction in epimerase activitytowards partially desulfated DS; (ii) heterologous expression ofmutant full-length and soluble recombinant DSE proteins inCOS-7 cells show an activity decreased to mock control levelstowards partially desulfated DS despite wild-type expressionlevels; (iii) we revealed decreased cellular and secreted DSlevels, similar to changes seen in D4ST1 deficiency, and an in-crease in cellular CS levels in the patient’s fibroblasts; and (iv)stable transfection-related expression of wild-type DSE increasedthe amount of DS disaccharide secreted by patient fibroblasts. Ourwork reveals locus heterogeneity in MCEDS, and shows that DSEand D4ST1 act in concert to form IdoUA stretches in chondroitin,thereby producing mixed CS/DS and DS GAG chains, in a largenumber of human connective tissues. DSE deficiency is likelyto affect many types of proteoglycans, which might explain thecomplex phenotype.

RESULTS

Identification of a DSE missense mutation in a MCEDSpatient without CHST14 mutation

A diagnosis of MCEDS was made in a cognitively normal boy at2 years of age based on the presence of characteristic facial fea-tures, congenital contractures of the thumbs and the feet, hyper-mobility of finger, elbow, and knee joints and a tendency toatrophic scarring of the skin (Fig. 2). The finding of muscleweakness suggested a myopathy in the patient.

Sequence analysis of CHST14, the gene found to be mutated inall 23 MCEDS patients reported to-date, revealed no mutations.Considering locus heterogeneity in MCEDS, we next deter-mined the localization of homozygous genomic regions in thisoffspring of consanguineous parents by high-density SNParray genotyping. We found that 19.54% of the patient’sgenome was homozygous, with 25 homozygous regions exceed-ing 4 Mb in size (data not shown). CHST14 did not localizewithin a homozygous region, further excluding this gene ascausing the disease in our patient. We next selected DSE, local-izing to the largest homozygous region (44.689 Mb in size), onthe basis of its known function in DS biosynthesis as a candidatefor mutation analysis. We identified a homozygous DSE mis-sense mutation, c.803C.T (p.S268L, Supplementary Material,Fig. S1), in the patient. Both parents and a healthy brother were

Figure 2. Clinical features of MCEDS due to DSE deficiency. Indian patienthomozygous for DSE mutation p.S268L at age 2 years. Note characteristicfacial features (see text), brachycephaly, long and tapering fingers, excessivelywrinkled palms and clubfeet.

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heterozygous for this mutation. This mutation is predicted byPolyPhen-2 to probably damage the protein function (HumVarscore of 0.994), and was absent from the 1000 genomes,dbSNP and the NHLBI Exome Sequencing Project databases(queried on 2013-03-07), as well as from 300 Caucasianin-house control samples. Serine-268 in DSE was found to rep-resent a highly conserved residue in DSE orthologs and in humanDSEL (Supplementary Material, Fig. S2).

DSE had been originally cloned as a gene encoding a squamouscell carcinoma antigen recognized by cells of the HLA-A24-restricted and tumor-specific cytotoxic T-lymphocyte line; thegene was named SART2 (squamous cell carcinoma antigenrecognized by T cells 2) but not assigned any specific functionat the time (42). In 2006, the SART2 protein was independentlypurified from bovine spleen microsomes, identified by mass spec-trometry, and shown to convey DS epimerase activity (15). DSEencodes a protein of 958 amino acids, which shares a conserved�700 amino acid domain with DSEL; significant homology isfurther observed with bacterial alginate oligo-lyases (UniProtKBaccession numbers 3A0O, 3AFL). Lyase and epimerase reactionsare mechanistically related and are at times expressed by a singleprotein (e.g. alginate AlgE7) (43). No sequence similarity isobserved with HS epimerases (14). Consistent with the role of aGAG-modifying enzyme, and the predictions of an N-terminalendoplasmic reticulum (ER) membrane retention signal and anN-terminal transmembrane domain, subcellular localizationanalyses of tagged DSE and immunoprecipitation experimentsindicated localization in ERs and in part in Golgi. DS epimeraseactivity is ubiquitously present in normal tissues, although withquantitative differences (15). In mice, DSE contributes to themajor part of enzyme activity in extracts of most tissues, withthe lowest expression in the brain (44).

The identified DSE mutation causes loss of epimerase activity

We hypothesized that the DSE mutation p.S268L compromisedepimerase function and caused MCEDS in our patient. This wasinvestigated by assaying epimerase activity towards partiallydesulfated DS as an acceptor in patient and control fibroblasts,and in COS-7 cells transiently over-expressing full-length andsoluble recombinant mutant and wild-type enzymes, respectively.

Our strategy for the characterization of DSE reaction productsis shown in Supplementary Material, Figure S3, and the substratespecificity of bacterial chondroitinases used in this study isshown in Supplementary Material, Figure S4. DSE catalyzesthe epimerization of GlcUA into IdoUA in chondroitin chainsand the reverse reaction in dermatan chains. Microsomal DSEpreparations from fibroblasts showed a higher epimerase activitytoward the carboxy group of IdoUA than GlcUA in vitro (45),i.e. the reverse reaction is favored. Thus, extrinsic dermatanrather than chondroitin was utilized as substrate for the DSEassay.

Heterologous expression of mutant full-length recombinantDSE proteins showed an activity decreased to mock controllevels towards partially desulfated DS despite wild-type expres-sion levels (Fig. 3).A soluble form of DSE was created by deletingthe first 31 amino- as well as the 58 carboxy-terminal amino acids.Consistently, the mean activity of the soluble mutant form of theenzyme was significantly decreased to mock control levels, instrong contrast to the wild-type enzyme (Supplementary Material,

Fig. S5). Furthermore, the cell lysate from the patient-derivedfibroblasts demonstrated a significant reduction in epimerase ac-tivity (Fig. 4). These results suggest that the DSE mutationp.S268L causes loss of epimerase activity, resulting in aberrantsynthesis of DS chains and DS-PGs.

The DSE mutation p.S268L causes a decrease in DSbiosynthesis and leads to an increase in cellular CS chains

We compared the composition of CS/DS chains from patient andcontrol fibroblasts in order to determine whether the DS biosyn-thesis was disturbed. In three sets of experiments, the GAG frac-tions from conditioned media and fibroblast lysates were digestedwith different chondroitinases to determine the amounts of DSmoieties (Fig. 5 and Supplementary Material, Table S5), theamounts of CS moieties (Supplementary Material, Fig. S7 andTable S4) and CS and DS moieties together (Supplementary Ma-terial, Fig. S6 and Table S3). The CS/DS-derived disaccharideswere fluorophore labeled and separated by anion-exchangeHPLC, and the amount of disaccharides in each sample was calcu-lated from the peak area in the chromatogram.

The results of all GAG measurements are summarized inTable 1. In accordance with an epimerase deficiency in mutantfibroblasts, we found a reduction in the IdoUA content of theCS/DS chains from the cell and culture medium fractions of thepatient fibroblasts. The amount of DS disaccharides from the con-ditioned media was reduced to �10% in the patient when com-pared with the control, whereas no apparent difference wasobserved in the amount of CS chains (Table 1). The amount ofDS disaccharides obtained from the cell fraction was alsoreduced to �20%, while CS-derived disaccharides was increasedby �1.5 times compared with the control cells (Table 1). This ob-servation suggests that a reduction in the DS biosynthesis causedby the mutant DSE resulted in an increased synthesis of CS chainsin the cell fraction or an accumulation of CS chains, which couldnot be converted to DS chains.

Our finding of a �1.5 times larger amount of disaccharidesliberated by chondroitinase ABC digest, corresponding to thetotal release of GlcUA and IdoUA linkages present, comparedwith the sum of separate releases of CS and DS disaccharidesfrom the cell fraction indicated the presence of large amountsof otherwise rare linkage patterns, which were resistant tothese enzymes. However, no significant difference was observedbetween the patient and the control with regard to the amount oflarger oligosaccharides upon gel-filtration chromatography ofthe chondroitinase B digests of CS/DS chains of the cell and con-ditioned medium fractions from the fibroblasts (data not shown).Thus, the mutation of DSE results in the reduction in IdoUA clus-ters rather than sparsely distributed IdoUA residues in DS andCS/DS hybrid chains as was demonstrated in Dse null mice (46).

The content of the IdoUA-GalNAc(4-O-sulfate)- structurecharacteristic for DS on decorin is reduced in the patient

We examined whether DSE deficiency affected the CS/DS hybridchain on decorin, a major DS proteoglycan in the skin. The CS/DShybrid chain on decorin is normally composed of a DS moiety(�95% as disaccharides) and a CS moiety (�5% as disacchar-ides) (27). A western blotting was performed for decorin coreprotein after digestion of the CS/DS hybrid chain with the













chondroitinases ABC, AC-I and B. The CS/DS hybrid chain ondecorin was susceptible to all three types of chondroitinases,giving a single protein band in each case, which suggested thatthe side chain was of a CS/DS hybrid type. However, the molecu-lar size of the bandobtained using chondroitinaseB appeared to beslightly larger, and that of the intact decorin proteoglycan muchlarger than that of the control (Fig. 6). This suggests that theGAG is larger than normal, and the cleavage site in the glycan

Figure 3. Expression and assays of the DSE activity of the full-length form of therecombinant DSE proteins (wild-type and p.S268L). (A) Western blotting of therecombinant DSE-V5 (wild-type and S268L). The recombinant DSE (wild-typeand p.S268L) was transiently expressed in COS-7 cells, separated by 7.5%SDS–polyacrylamide gel electrophoresis, and detected with anti-V5 antibody.‘Empty’ indicates the cell lysate from COS-7 cells transfected with an emptyvector. (B) Comparison of the DSE activity of the full-length form of recombinantDSE (mean+SE, n ¼ 3). Enzyme assays were carried out as described in Materi-als and Methods. The strategy for the characterization of DSE reaction products isdescribed in the legend to Supplementary Material, Figure S3. Briefly, the recom-binantDSE (wild-typeormutant) was incubated with partially desulfated DSas thesubstrate. The DSE converts the IdoUA to GlcUA by its reverse-epimerizing activ-ity, resulting in the formation of GlcUA residues, which are substituted by GalNAcresidues. These linkages become susceptible to chondroitinase AC (Supplemen-tary Material, Figs S3 and S4). After the incubation of the reaction products withchondroitinase AC, the resultant di- and oligo-saccharides such as DHexUA-GalNAc, DHexUA-GalNAc-IdoUA-GalNAc, DHexUA-GalNAc-IdoUA-GalNAc-IdoUA-GalNAc etc. were yielded. Each saccharide was labeled with afluorophore (2-aminobenzamide) at the reducing terminus, and separated byanion-exchange HPLC (C–F). The DHexUA (4,5-unsaturated hexuronic acid)residue should be originally derived from a GlcUA residue due to the susceptibilityto chondroitinase AC. Consequently, each saccharide contains a DHexUA at non-reducing end, and was utilized for quantifying the GlcUA content (SupplementaryMaterial, Tables S6 and S7). The sum of each saccharide was described in the (B)with bar graphs. ‘Mock’ and ‘empty’ indicate the DSE activity from cell lysates ofCOS-7 cells transfected without and with an empty vector, respectively. ∗P ,0.0001 by Student’s t-test. (C–F) Anion-exchange HPLC of the DSE reaction pro-ducts prepared using the full-length form of recombinant DSE of wild-type (E) andp.S268L (F) and partially desulfated DS as substrate. Representative chromato-grams from each reaction product are shown in the figure. (C )and (D) show the

chromatograms of the substrate control and the reaction products of the celllysate transfected with an empty vector, respectively. The DSE reaction productswere digested with a mixture of chondroitinases AC-I and AC-II into di- and oligo-saccharides, labeled with 2AB, and the 2AB-labeled oligosaccharides were sepa-rated by anion-exchange HPLC on an amine-bound silica PA-G column using alinear gradient of NaH2PO4 as indicated by the dashed line. The elution positionsof authentic 2-AB-labeled di- and oligosaccharides derived from partiallydesulfated DS [Mikami et al. (21)] are indicated by numbered arrows: 1, DHexUA-GalNAc; 2, DHexUA-GalNAc-IdoUA-GalNAc; 3, DHexUA-GalNAc-IdoUA-GalNAc-IdoUA-GalNAc; 4, DHexUA-GalNAc-IdoUA-GalNAc(4S); 5, DHexUA-GalNAc-IdoUA-GalNAc-IdoUA-GalNAc(4S)/DHexUA-GalNAc-IdoUA-GalNAc(4S)-IdoUA-GalNAc; 6, DHexUA-GalNAc(4S)-IdoUA-GalNAc(4S); 7,DHexUA-GalNAc-IdoUA-GalNAc(4S)-IdoUA-GalNAc(4S); 8, 4-O-disulfatedoctasaccharide.

Figure 4. DSE activity in the skin fibroblasts from a healthy control and thepatient. (A) Comparison of the DSE activity of the fibroblasts from a healthycontrol and the patient (mean+SE, n ¼ 3). The DSE reaction products wereanalyzed by anion-exchange HPLC (B–D). ∗P , 0.0005 by Student’s t-test.For the strategy of quantification of the DSE activity, see the legend toFigure 3. (B–D) Anion exchange HPLC of the DSE reaction products preparedusing the cell lysate from the control (C) and the patient (D) and partially desul-fated DS as substrate. Representative chromatograms from each reaction productare shown in the figure. (B) shows the chromatograms of the substrate control.The DSE reaction products were digested with a mixture of chondroitinasesAC-I and AC-II into di- and oligosaccharides, labeled with 2AB, and the2AB-labeled oligosaccharides were separated by anion-exchange HPLC asdescribed in the legend to Figure 3. For the elution positions of authentic2-AB-labeled di- and oligosaccharides, see the legend to Figure 3.








chainforchondroitinase Bis moredistant to the glycanattachmentsite on the core protein than that of the cleavage sites for the othertwo enzymes. Although not unambiguously demonstrated bydecorin western blotting, our findings of a reduced IdoUAcontent of cellular and secreted GAGs suggest that the contentof the IdoUA-GalNAc(4-O-sulfate)- structure characteristic forDS on decorin is reduced in the patient.

Rescue experiments performed by stable expressionof wild-type DSE in patient fibroblasts increased the amountof secreted DS disaccharides

Stable expression vectors, pEBMulti-empty and -DSE (wild-type), were transfected into the patient cells, and the cells werepropagated in the presence of G418. An analysis of DS chainswas performed after digestion with chondroitinase B. Theamount of DS disaccharides, DHexUA-GalNAc(4S), secretedby fibroblasts stably expressing DSE increased 3-fold comparedwith that obtained from cells transfected with the empty vector(Supplementary Material, Table S8 and Fig. S8). These observa-tions strongly suggest that the patient shows a loss of functionof DSE.

Taken together, our data indicate that the p.S268L mutationaffects the epimerase activity, resulting in a decreased IdoUAcontent of DS chains, i.e. a reduced DS biosynthesis, and in anincreased synthesis or an accumulation or reduced conversionof CS chains in the cell fraction, which cannot be converted toDS or hybrid CS/DS chains.

DISCUSSION

CHST14 mutations were recently identified in two distinct seriesof patients clinically diagnosed with a supposedly new form ofEDS, named EDSKT (27), and with ATCS, and shown toresult in D4ST1 deficiency (26). A comprehensive review ofpatients with D4ST1 deficiency reported to-date, variably diag-nosed as ATCS, EDSKT and MCEDS, supports the notion thatD4ST1 deficiency represents a single clinical entity, with some

Figure 5. HPLC profiles of the digests of the GAG fractions prepared from theconditioned media (A and B) and cell fractions (C and D) of the fibroblast culturesafter treatment with chondroitinase B. The GAG fractions containing CS/DSchains from a healthy control (A and C) and the affected individual (B and D)were digested with chondroitinase B, which specifically acts on the IdoUA-containing structure in DS or CS/DS hybrid chains, into disaccharides, labeledwith 2AB, and 2AB-labeled CS/DS disaccharides were separated byanion-exchange HPLC on an amine-bound silica column using a linear gradientof NaH2PO4 as indicated by the dashed line. The elution positions of authentic2-AB-labeled CS disaccharides are indicated by numbered arrows: 1, DHexUA-GalNAc; 2,DHexUA-GalNAc(6S); 3,DHexUA-GalNAc(4S); 4,DHexUA(2S)-GalNAc(6S); 5, DHexUA(2S)-GalNAc(4S); 6, DHexUA-GalNAc(4S,6S); 7,DHexUA(2S)-GalNAc(4S,6S).

Table 1. The total amount of fibroblast-derived GAGs from CS/DS chains

CS/DSa CSb DSc Hyaluronand

Conditioned medium nmol/mg proteinControl 47.9 29.9 18.3 68.5Patient 16.2 20.8 1.7 85.3

Cell fraction nmol/mg proteinControl 1.2 0.90 0.33 7.2Patient 2.2 1.4 0.066 4.1

Values, which showed clear or characteristic differences between the control andpatient samples, are highlighted in bold.aTotal amount of disaccharides derived from CS/DS chains were calculatedbased on the peak area in the chromatograms of the digests with a mixture ofchondroitinases ABC and AC-II (Supplementary Material, Fig. S6).bTotal amount of disaccharides derived from the CS moiety of CS/DS chainswere calculated based on the peak area in the chromatograms of the digests with amixture of chondroitinases AC-I and AC-II (Supplementary Material, Fig. S7).cTotal amount of disaccharides derived from the DS moiety of CS/DS chainswere calculated based on the peak area in the chromatograms of the digests withchondroitinase B (Fig. 5).dTotal amount of disaccharides derived from hyaluronan were calculated basedon the peak area in the chromatograms of the digests with a mixture ofchondroitinases AC-I and AC-II (Supplementary Material, Fig. S7).

Figure 6. Composition of the CS/DS hybrid chain on decorin. A western blot wasperformed for decorin core protein after digestion of the CS/DS hybrid chain withthe chondroitinases ABC, AC-I and B revealing that the side chain is of a CS/DShybrid type. However, the molecular size of the band obtained using chondroiti-nase B may appears to be slightly larger, and that of the intact decorin proteogly-can is much larger than that of the control.







degree of variability observed in inter- and intra-familial diseaseexpression (28,36–38).

The disorder, MCEDS, is characterized by progressive multi-system fragility-related manifestations including joint disloca-tions and deformities, skin hyperextensibility, bruisability andfragility; recurrent large subcutaneous hematomas, and othercardiac valvular, respiratory, gastrointestinal, and ocular com-plications, which are considered to result from connectivetissue weakness. The disorder also shows various developmentalmalformations including distinct craniofacial features, multiplecongenital contractures and congenital defects in cardiovascu-lar, gastrointestinal, renal, ocular and central nervous systems.MCEDS also affects muscles to a variable degree (38).MCEDS shares many clinical features with the kyphoscoliotictype of EDS (47), the tenascin-X deficient EDS (48),FKBP14-deficient EDS (41) and collagen VI-related Ullrichcongenital muscular dystrophy (UCMD [MIM 254090]) andBethlem myopathy (MIM 158810) (49). In addition, clinicaloverlap can be found at birth with distal arthrogryposis type 9(congenital contractural arachnodactyly, CCA, Beals syndrome[MIM 121050]) due to FBN2 (MIM 612570) mutations and theLoeys-Dietz syndrome (LDS [MIM 609192]) within the firstyears of life. MCEDS can be diagnosed at birth by the presenceof characteristic craniofacial features—consisting of brachy-cephaly, hypertelorism, down-slanting palpebral fissures,small mouth with thin vermillion border, prominent cheeks,microretrognathia and protruding ears—and distal arthrogrypo-sis, and molecular genetic testing of CHST14 was so far consid-ered to provide a definitive diagnosis.

The patient we describe here presented with characteristic fea-tures of MCEDS, and we consequently show that there is locusheterogeneity in MCEDS: direct and indirect DNA analysiswidely excluded CHST14 as disease-causing in our patient,and the targeted analysis of a single candidate gene, DSE, froma linkage interval identified a missense mutation affecting ahighly conserved residue. We present several lines of evidencethat this mutation affects the DS eperimase’s function. Weshow that the recombinant mutant enzyme’s activity, as wellas the patient-derived fibroblast’s epimerase activity towardspartly desulfated DS, is severely decreased. We observedecreased amounts of IdoUA-GalNAc(4-O-sulfate) linkagesand increased amounts of GlcUA-GalNAc(4-O-sulfate) andGlcUA-GalNAc(6-O-sulfate) in the cellular and secreted GAGfractions of patient’s fibroblasts after specific chondroitinasedigests. We show that residual IdoUA-GalNAc(4-O-sulfate) lin-kages are present and unusually scattered over hybrid CS/DSchains. Fibroblasts with the p.S268L mutation in DSE epimerizeGlcUA-GalNAc to IdoUA-GalNAc at a reduced rate resulting insecretion of about 10% IdoUA-containing disaccharides com-pared with a control. A significant fraction of DS is replacedby CS, which cannot generally substitute functions specificallyassociated with DS, as both D4ST1 and DSE deficiency causesMCEDS. Finally, we suggestively rescue the biochemicalphenotype by stable expression of wild-type DSE in patientfibroblasts.

Conversion of GlcUA to IdoUA by DS epimerases is a freelyreversible reaction that favors the GlcUA form, and the additionof sulfate to the C4 hydroxy of GalNAc adjacent to IdoUA byD4ST1 prevents further back epimerization of IdoUA toGlcUA (21,45). The notion of a spacio- and temporally

coupled action of DSE and D4ST1 in the generation of IdoUAdisaccharides and IdoUA blocks is supported by our reportingof MCEDS phenotypes resulting from deficiencies of eitherDSE or D4ST1, and both causing shortages of IdoUA residuesin CS/DS chains in a number of connective tissues.

Apparently, DSELcannotcompensate for the lossof DSE func-tion, analogous to the situation where CS 4-O-sulfotransferasescannot compensate for the lossof D4ST1, and in linewith reportedinvitro substrate specificities of human sulfotransferases involvedin the 4-O-sulfation of CS/DS (20,21).

MCEDS might result both from abnormal and from loss ofDS-proteoglycan functions. The structure of DS chains is moreflexible than that of CS (50,51), which might affect DS proteo-glycans such as decorin. Decorin directly binds to collagen viaits core protein, and the GAG side chains aggregate and functionas interfibrillar bridges (52,53). The observed connective tissueweakness in MCEDS patients might represent a direct conse-quence of insufficient decorin-mediated assembly of collagenfibrils caused by either D4ST1 or DSE deficiency. Indeed, weshowed previously that the CS/DS side chain was replaced bya CS chain on the decorin molecule in skin fibroblasts fromD4ST1-deficient patients and no DS moiety was detected (27).Similarly, our current study indicates that a deficiency ofIdoUA residues in the decorin CS/DS chain results from DSE de-ficiency. Transition from the CS/DS hybrid chain of decorin to aCS chain probably decreases the flexibility of the GAG chain.Unfortunately, no material for electron microscopy of the skinis available from our patient. The rare studies of skin specimenfrom D4ST1-deficient patients showed that the size and shapeof skin collagen fibrils were normal, but the collagen bundlesappeared progressively and abnormally dispersed in the reticulardermis. The observation that dermal collagen fibrils showedhuge varieties of size and shape in decorin null mice implicatedthe core protein of decorin as important for collagen fibril forma-tion (54), and our findings suggest that the CS/DS hybrid chain ofdecorin regulates the space between the collagen fibrils andforms collagen bundles as indicated previously (27,55). Thisnotion is supported by observing similar alterations, i.e. fibrilswith larger diameter, in Dse null mice and in D4ST1-deficientskin when compared with controls (27,46). In line with our find-ings in the DSE-deficient patient, the amount of long IdoUAblocks was shown to be greatly decreased in decorin, biglycanand versican-derived CS/DS chains in Dse null mice (46). Dse-deficient mice were smaller than their wild-type littermates. AllDse null pups had a kinked tail that was not present after 4 weeksof age. Dse null mice showed otherwise no gross macroscopicalterations, but a reduced fertility (46). Dsel and Dse areexpressed in all tissues in mice; Dse was the predominant epi-merase in three of five tissues examined, i.e. the skin, lung andspleen, and the brain is the only tissue where most of the activity(89%) was attributed to Dsel (56,57). DSEL might encode thepredominant epimerase in human brain as well, correlatingwith the absence of cognitive constraint in the patient. Theamount of IdoUA in CS/DS proteoglycans varies betweentissues and depends on the developmental stage (58,59);genetic and environmental factors, which contribute to its regu-lation, might explain some of the phenotypic variabilityobserved between MCEDS patients.

Hypomorphic mutations in two enzymes involved in the syn-thesis of the GAG linker tetrasaccharide, encoded by B4GALT7



(MIM 604327) and B3GAT3 (MIM 606374), result in deficien-cies of more than one class of GAG chains, and cause theprogeroid form of EDS (60–62) and a connective tissue disorderwith multiple joint dislocations, short stature, craniofacial dys-morphism and congenital heart defects (63), respectively.MCEDS shares joint hypermobility with both conditions, andsome facial features and an increased incidence of heartdefects with the latter disorder, and this overlap might resultfrom a reduction in the amount of DS proteoglycans.

The phenotypic overlap of MCEDS and the tenascin-X (TNX[MIM 600985])-deficient form of EDS (MIM 606408) (64)might be caused by a disruption of extracellular binding ofdecorin and tenascin-X, which also binds to tropoelastin and col-lagen types I, III, V, XII and XIV (65,66). In contrast to D4ST1deficiency, where ecchymoses and hematoma formation arecommon (37), this was not observed in our young patient withDSE deficiency so far. A deficiency of DS is supposed toaffect DS-mediated activation of heparin cofactor II (HCF2[MIM 142360]) in the arterial wall after endothelial injury (67).

Quantitative and qualitative changes in the DS content oftissues are supposed to have effects on the generation of morpho-gen gradients in epithelia (5,6,68). For example, it has beenshown that decorin neutralizes the activity of TGFB1 (69), anddeficiency or substitution of DS chains by CS on decorinwould implicate altered TGFB signaling in the pathogenesis ofMCEDS, as it was shown in LDS (70) and Sphrintzen-Goldbergsyndrome (SGS). Altered TGFB signaling might be responsiblefor features which are shared by LDS, SGS and MCEDS such as aMarfanoid habitus, hypertelorism, down-slanting palpebralfissures, arachnodactyly, joint hypermobility and jointcontractures (71,72).

Of note, a DSE upregulation resulting in 5-fold increase in theCS/DS content and changes in growth factor binding and subse-quently in pErk1/2 signaling have been found in esophagus squa-mous carcinoma, an aggressive tumor with poor prognosis,indicating that IdoUA in DS influences tumorigenesis by affect-ing cancer cell behavior (73). Upregulation of DSE has beenobserved in a number of other types of cancer as well (42).

We speculate that the variety of symptoms observed inMCEDS is caused by the deficiency of decorin and further DSproteoglycans, of which there are currently 29 known but lesswell studied than decorin, as well as due to the lack of DS inthe vascular endothelia. The DSE and D4ST1 deficiencies repre-sent disorders that specifically affect the DS biosynthesis, andemphasize roles of this GAG in human development and extra-cellular matrix maintenance (74).

MATERIALS AND METHODS

Patient and family

A 2-year-old Indian boy, born to healthy, consanguineousparents (first degree cousins), was clinically diagnosed withMCEDS based on the presence of facial dysmorphism, consist-ing of frontal bossing, open anterior fontanelle, downward-slanting palpebral fissures, telecanthus, bluish sclerae, higharched palate, tent-shaped lips, dental crowding, brachycephalyand prominent ears, as well as arachnodactyly, adducted thumbs,joint hyperlaxity, inguinal hernia and congenital bilateral talipesequino varus. Cardiac ultrasonography showed a patent foramen

ovale, and a computed tomography of the brain showed a gener-alized mild cerebral atrophy. Following clubfoot surgery, thepatient showed delayed wound healing and atrophic scarringof the skin. Generalized muscle weakness was observed pointingto an underlying myopathy. However, a muscle biopsy was notobtained. While his gross motor development was delayed, hiscognitive development was normal.

SNP array hybridization, homozygosity mappingand DNA sequence analyses

Written informed consent for molecular studies was obtainedfrom the parents, and the study was conducted in accordancewith the principles of the Declaration of Helsinki. GenomicDNA was isolated from peripheral blood leukocytes by standardprocedures using a robot (BioRobot M48, QIAGEN). A genome-wide homozygosity scan and copy-number variant detection (mo-lecular karyotyping) were performed following hybridization ofthe patient’s DNA sample to a HumanCytoSNP-12v2 BeadChipSNP array (Illumina) interrogating 299 140 markers, and accord-ing to the manufacturer’s instructions. Raw SNP call data wereprocessed with the Genotyping Analysis Module of GenomeStu-dio 1.6.3 (Illumina). Copy-number variants and segments of lossof heterozygosity were called and visualized using Nexus soft-ware and the SNPFASST segmentation algorithm (BioDiscoveryInc.). A parametric multipoint logarithm of the odds score calcu-lation was performed with the ALLEGRO program (75) using anautosomal recessive, fully penetrant model and marker allele fre-quencies determined out of 250 individuals of European descentfrom our in-house array facility.

The coding regions and splice sites of the CHST14 and DSEgenes were PCR amplified and directly sequenced in ourpatient. Primer sequences were based on the NCBI referenceentry for CHST14 mRNA (NM_130468.3) and two availableNCBI mRNA reference entries for DSE transcripts, whichdiffer in their untranslated first exons (NM_001080976.1 andNM_013352.2). GoTaq polymerase and buffer (Promega,Mannheim, Germany) were used to PCR-amplify 20–50 ng ofgenomic DNA in 35 cycles of 20 s at 958C, 20 s at 608C and30 s at 728C in addition to an 7 min final extension, applied toall sets of primers (Supplementary Material, Table S1). Theamplicons were cleaned using ExoSap-IT (USB, Vienna,Austria), and subsequently sequenced using an M13 universalprimer on an ABI 3730s automated sequencer, with BigDye ter-minator mix (Applera, Vienna, Austria). Sequence chromato-graphs were analyzed using the Sequence Pilot computerprogram (JSI Medical Systems, Kippenheim, Germany). Theparents and a healthy brother were tested for the mutationdetected in the patient by sequence analysis. A panel of 300 Cau-casian DNA samples from anonymous controls was analyzed forthe presence of the identified DSE mutation by an allele-specificPCR (primers are shown in Supplementary Material, Table S2).

Primary fibroblast cultures

We obtained skin fibroblasts from the patient at age 3 years, andan age-adjusted control. Cells were cultured in Dulbecco’smodified Eagle’s medium with 10% heat-inactivated fetalbovine serum, 100 U/ml penicillin, 100 U/ml streptomycin and2 mM L-glutamine (Invitrogen).





Construction of the expression vectors of the wild-typeDSE and the p.S268L mutant protein

The expression vector of the full-length open reading frame en-coding human DSE (wild-type) was amplified by PCR using thespecific primers (5′-GGG GTA CCG CCA CGA TGA GGACT-3′ and 5′-GGA CTA GTA CAC TGT GAT TGG GA-3′),pBluescriptR/human DSE (IMAGE clone #5272885, Open Bio-systems, Huntsville, AL, USA) as a template, and KOD-PlusDNA polymerase (Toyobo, Tokyo, Japan) described previouslywith a slight modification (15). The amplified fragments weredigested with KpnI and SpeI, and subcloned into of pEF6/V5-His-B (Invitrogen). The resultant expression vector, pEF6/hDSE (wild-type)-V5-His, was digested with KpnI and PmeI,and the cDNA fragments containing the open reading frame ofDSE were inserted into the expression vector, pEBMulti-Neo(Wako, Osaka, Japan).

In addition, the expression vector containing a soluble form ofDSE was also constructed as described previously (14). Briefly, atruncated form of DSE (wild-type), lacking the first 31 amino-and 58 carboxy-terminal amino acids, respectively, of theDSE, was amplified by PCR using a 5′-primer containing ClaIsite (5′-CCA TCG ATA ATG ATT CCC TTC ACC-3′) and a3′-primer containing a KpnI site (5′-GGG GTA CCG AATAGG AAG CAG ACA G-3′) and pBluescriptR/human DSE asa template. The resultant DNA fragments were inserted intop3xFLAG-CMV13 (Sigma).

The vector of the mutant protein, S268L, was constructedaccording to the manufacturer’s instructions using the Quick-Change II Site-Directed Mutagenesis Kit (Stratagene) and theprimers (5′-AGC TAC ACC ACT AGA TTA CTC TTC CAATAC ATG-3′ and 5′-CAT GTA TTG GAA GAG TAA TCTAGT GGT GTA GCT-3′), in which the underlined nucleotidesindicate the point mutations.

Expression and western blotting of the recombinant enzymes

The expression plasmids (3 mg) were transiently transfected intoCOS-7 cells (Japanese Collection of Research BioresourcesCell Bank, Osaka, Japan) in a culture dish (100 mm in diameter)using 10 ml of FuGENETM HD transfection reagent (Promega).After 3 days, the cells transfected with pEBMulti/hDSE-V5 werelysed with 200 ml of 25 mM 2-morpholinoethanesulfonic acid(MES)-NaOH, pH5.5, containing 10 mM MnCl2 and 0.25%NP-40.

On the other hand, after 3 days the culture medium (2 ml) fromCOS-7 cells transfected with p3xFLAG-CMV13/hDSE-FLAGwas incubated with 15 ml of anti-FLAG affinity agarose resin(Wako) at 48C overnight, the enzyme-bound affinity resin waswashed with 25 mM Tris–HCl, pH 7.4, containing 150 mM

NaCl and 0.05% Tween-20, and subjected to sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (SDS–PAGE). Western blotting was carried out using the horseradishperoxidase-conjugated anti-FLAG monoclonal antibody andthe ImmunoStar LD detection Kit (Wako). The signals of thechemi-luminescence were detected by LAS-4000mini (Fuji-Film, Tokyo, Japan).

Determination of the DSE activity

DSE activity was assayed by the method described previously(14,15,45) with a modification summarized in SupplementaryMaterial, Figure S3. Briefly, the enzyme-bound anti-FLAGresin (15 ml) or cell lysate (20 ml) as an enzyme source, partiallydesulfated DS (9 nmoles as disaccharides), which was preparedpreviously (21), as the acceptor substrate were mixed in a finalvolume of 30 ml of 25 mM MES-NaOH, pH 5.5, containing10 mM MnCl2 and 0.25% NP-40. The assay mixtures were incu-bated overnight at 378C. An aliquot of the reaction productswere individually incubated with a mixture of chondroitinasesAC-I and AC-II (EC 4.2.2.5) (Seikagaku Biobusiness, Tokyo,Japan) in a total volume of 20 ml of 50 mM Tris–HCl, pH 8.0, con-taining 60 mM CH3COONa at 378C for 60 min (76). Reactionswere terminated by boiling for 5 min. Each digest was labeledwith a fluorophore, 2-aminobenzamide (2AB),as described previ-ously (77). Separation and quantification of 2AB-derivatized oli-gosaccharides were carried out as described previously with aslight modification (77). Briefly, the 2AB-labeled oligosacchar-ides were subjected to anion-exchange HPLC on an amine-boundsilica PA-G column (4.6 × 150 mm, YMC Co., Kyoto, Japan)connected with a guard column, and eluted with 16 mM

NaH2PO4 for 10 min followed by a linear gradient of NaH2PO4

from 16 to 530 mM over 60 min and then eluted with 1 M

NaH2PO4 for 5 min at a flow rate of 0.5 ml/min at room tempera-ture. The eluates were monitored using an RF-10AXL fluoromet-ric detector (Shimadzu Co., Kyoto, Japan) with excitation andemission wavelengths of 330 and 420 nm, respectively. Eacholigosaccharide was identified by comparison of the retentiontime of the structure-defined oligosaccharide standards (21).The amount of di- and oligosaccharides in each sample was calcu-lated on the basis of the peak area in the chromatogram. DSE ac-tivity as conversion of IdoUA to GlcUA was determined by thetotal amount of each oligosaccharide yielded by the treatment ofthe reaction products with chondroitinase AC.

Disaccharide and oligosaccharide composition analysisof CS/DS chains isolated from fibroblasts

After incubation for 2 days, the medium was replaced to serum-free medium for fibroblasts (Cosmobio H001, Tokyo, Japan).The medium was collected after 3 days, concentrated (AmiconUltra-4 (10k) (Millipore) and a proteinase inhibitor cocktail(Nacalai tesque, Kyoto, Japan) was added. Fibroblasts culturedfor 3 days on 150 mm plates were collected using a scraper(BD Falcon), dried with acetone and GAGs were precipitatedwith 80% ethanol and sodium acetate as described (76). Afterbeing desalted using a centrifugal filter (Amicon Ultra-4;Ultracel-3k, Millipore Corp., Billerica, MA, USA), thesamples were treated individually with a mixture of chondroiti-nases ABC (EC 4.2.2.20) and AC-II (EC 4.2.2.5) (SeikagakuCorp., Tokyo, Japan), a mixture of chondroitinases AC-I (EC4.2.2.5) and AC-II (Seikagaku Corp.), or chondroitinase B (EC4.2.2.19) (IBEX Technologies, Montreal, Canada) for analyzingthe disaccharide composition of CS/DS, CS or DS, respectively.All three chondroitinases generate unsaturated uronic acid(DUA)-containing disaccharides. Chondroitinase AC degrades





CS, chondroitinase B degrades DS and chondroitinase ABCdegrade both CS and DS (Supplementary Material, Fig. S4).The digests were labeled with a fluorophore 2-aminobenzamide(2AB) and aliquots of the 2AB-derivatives of CS/DS oligosac-charides were analyzed by anion-exchange HPLC on a PA-03column (YMC Co., Kyoto, Japan) as previously described(77). The unsaturated CS/DS disaccharides observed in thedigests were identified by comparison with the elution positionsof authentic 2AB-labeled disaccharide standards. The amountsof di-, tetra-, hexa-, octa- and decasaccharides from chondroiti-nase B cleavage reactions were separated by gel/filtration chro-matography on a Superdex peptide column.

Immunoblotting for decorin

The conditioned media from fibroblast cultures were digestedand subjected to SDS–PAGE using a 10–20% SDS polyacryl-amide gradient gel. Separated proteins were transferred to aPVDF membrane, and incubated for 2 h at 378C. Decorin CS/DS proteoglycan from fibroblasts was detected using anti-human decorin antibody (clone 115402; R&D Systems,Minneapolis, MN, USA).

WEB RESOURCES

The URLs for data presented herein are as follows:National Center for Biotechnology Information [NCBI; for

data on DSE, CHST14 and their orthologs (RefSeq, Pubmed,OMIM, Gene, Protein)], and a BLAST homology search:http://www.ncbi.nlm.nih.gov/.

Databases of DNA sequence variants: 1000genomes database(http://www.1000genomes.org/), dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) and the NHLBI Exome SequencingProject (http://evs.gs.washington.edu/EVS/).

European Bioinformatics Institute Web site (for ClustalW),http://www.ebi.ac.uk/index.html.

PolyPhen-2: prediction of functional effect of human nsSNPs,http://genetics.bwh.harvard.edu/pph2/.

The OPAL protein domain and functional annotation analysistool at Oxford University, http://www.oppf.ox.ac.uk/opal/.

The UniProtKB protein structure database, http://www.uniprot.org/uniprot/?Query=reviewed%3Ayes.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We thank Ms Lyou Sunok, Hokkaido University, for her tech-nical assistance.

Conflict of Interest statement. None declared.

FUNDING

This work was supported by grants from Jubilaumsfonds derOsterreichischen Nationalbank (grant no. 14496 to T.M.), theMatching Program for Innovations in Future Drug Discovery

and Medical Care (to K.S.) from The Ministry of Education,Culture, Sports, Science and Technology, Japan (MEXT); aGrant-in-aid for Young Scientists (B) 23790066 (to S.M.)from the Japan Society for the Promotion of Science, Japan(JSPS); the Drs Hiroshi Irisawa and Aya Irisawa Memorial Re-search Grant from the Japan Heart Foundation (to S.M.); and aGrant-in-aid for Encouragement from the Akiyama LifeScience Foundation (to S.M.).

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