Molecular Basis for the Progeroid Variant of Ehlers-Danlos Syndrome

Progeroid type Ehlers-Danlos (E-D) syndrome was reported to be caused by defects in galactosyltransferase I (EC2.4.1.133), which is involved in the synthesis of common linkage regions of proteoglycans. Recently, we isolated cDNA of the galactosyltransferase I (XGalT-1) (Okajima, T., Yoshida, K., Kondo, T., and Furukawa, K. (1999) J. Biol. Chem.274, 22915–22918). Therefore, we analyzed mutations in this gene of a patient with progeroid type E-D syndrome by reverse transcription polymerase chain reaction and direct sequencing. Two changes of G and T to A and C at 186 and 206, respectively, were detected. Then, we determined the genomic DNA sequences encompassing the A186D and L206P mutations, revealing that the unaffected parents and two siblings were heterozygous for either one of the two different mutations and normal, while the patient had both of two different mutant genes. Enzymatic functions of cDNA clones of XGalT-1 containing the individual mutations were examined, elucidating that L206P clone completely lost the activity, while A186D retained ∼50% or 10% of the activity when analyzed with extracts from cDNA transfectant cells or recombinant soluble enzymes, respectively. Moreover, L206P enzyme showed diffuse staining in the cytoplasm of transfectant cells, while the wild type or A186D clones showed Golgi pattern. These results indicated that the mutations in XGalT-1 were at least one of main molecular basis for progeroid type E-D syndrome.

Glycosaminoglycans (GAGs) 1 are carbohydrate structures of different length, different types, and varying numbers on proteoglycan molecules (1). GAG synthesis is initiated by the transfer of xylose onto serine residues in core proteins. Sequential addition of two galactoses, and a glucuronic acid forms a common linkage structure detected in major proteoglycans. Alternative addition of GlcNAc or GalNAc residues to the common linkage structure leads to the formation of heparin/heparan sulfate or that of chondroitin sulfate/dermatan sulfate, respectively. Defects in the pathway of GAG synthesis should, therefore, cause serious abnormalities in a wide variety of tissues and organs, since proteoglycans are ubiquitously present and are thought to be involved in the regulation of cell proliferation/differentiation, tissue development and organogenesis (2), and infections (3).
Progeroid type Ehlers-Danlos (E-D) syndrome (OMIM 130070) was reported to be caused by defects in a glycosyltransferase, i.e. galactosyltransferase I (4) (EC 2.4.1.133), which is involved in the synthesis of common linkage regions of proteoglycans. Recently, we have isolated a cDNA of the galactosyltransferase I gene (XGalT-1) (5) from a cDNA library of a human melanoma cell line based on the search of expressed sequence tag data base. This gene showed high homology (38%) to Caenorhabditis elegans sqv-3 gene (6), and its product showed specific activity of galactosyltransferase upon p-nitrophenyl-␤-D-xylopyranoside with ␤1,4 linkage. Moreover, expression of the cDNA in the mutant CHO cells deficient of heparan sulfate expression resulted in the restoration of the expression of both heparan sulfate and chondroitin sulfate, indicating that the gene codes XGalT-1. Thus, availability of this gene enabled us to elucidate the molecular basis of progeroid type E-D syndrome.
In the present study, we report two different missense mutations in the XGalT-1 gene of a patient with progeroid type E-D syndrome, both of which were derived from his parents. We confirmed the functional defects of those cDNAs harboring individual mutations by introducing into XGalT-1 deficient mutant CHO cells. Moreover, we analyzed the alteration in the intracellular localization of the mutant enzymes. These results have clearly elucidated molecular basis for the defects in the GAG synthesis, and indicated the mechanisms for the pathogenesis of progeroid type E-D syndrome.

EXPERIMENTAL PROCEDURES
Patient and His Family-The clinical symptoms and signs of the patient with progeroid E-D syndrome were by Kresse et al. (7). Briefly, the patient exhibited an aged appearance, developmental delay, dwarfism, craniofacial dysproportion, generalized osteopenia, defective wound healing, hypermobile joints, hypotonic muscles, and loose but elastic skin. Enzyme assay of galactosyltransferase I revealed a marked reduction in galactosyltransferase I activity in the patient's fibroblasts (less than 1 ⁄20 of normal), and a moderate decrease in the parents (ϳ 1 ⁄2 of normal) (4).
Cell Culture-Mouse fibroblast L cells and Chinese hamster ovary cells (CHO-K1) were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 7.5% fetal calf serum at 37°C in a 5% CO 2 atmosphere. CHO mutant pgsB-761 (8) was obtained from the American Type Culture Collection and grown in F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum.
Sequence Analysis of XGalT-1 Gene-Established skin fibroblasts from the patient and his family were used as a source of RNA and DNA * This work was supported in part by Grants-in-aid for Center of Excellence research, for scientific research (10470029), and for priority areas (11139228, 10178104) from the Ministry of Education, Science, Sports and Culture and by the Ministry of Health and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Construction of Expression Vectors-Truncated form of XGalT-1 lacking 53 amino acids from the N terminus, was prepared by PCR using a 5Ј primer containing an EcoRI site, 5Ј-CAGCTCGAATTCTCTGGGGA-CGTGGCCCGG-3Ј, and a 3Ј primer containing a XhoI site, 5Ј-TGTCC-ACTCGAGTCAGCTGAATGTGCACCA-3Ј (nucleotides 1007-1024), and subcloned into EcoRI and XhoI sites of pCD-SA vector (kindly provided by Dr. Tsuji) as described (5). Full-length coding region of the cDNA was inserted at the down stream of a Myc epitope tag, which had been previously inserted into a mammalian expression vector pCDNA3 (Invitrogen).
Transfection-XGalT-1-deficient mutant CHO line pgsB-761 was transfected with wild type (WT), A186D and L206P cDNAs in pMIKneo expression vector (kindly presented by Dr. K. Maruyama at Tokyo Medical and Dental University) with LipofectAMINE TM (Life Technologies, Inc.) according to the manufacturer's instruction.
Western Blot Analysis-PgsB-761 cells transfected with 10 g each of Myc-tagged XGalT-1 and mutants were lysed in 100 mM MES buffer (pH 6.0) containing 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and total cell lysate (50 g) were separated by SDS-polyacrylamide gel electrophoresis and immuno-blotted with mouse anti-Myc tag antibody and biotinylated horse anti-mouse IgG. The antibody binding was revealed with ABC-PO (Vector) and HRP-1000 (Konica, Tokyo) according to the manufacturer's instruction.
Galactosyltransferase Assay-The galactosyltransferase activity was determined according to Lugemwa et al. (10) with modification. The assay mixture contained 2 l of Me 2 SO, 15 mM MnCl 2 , 50 mM KCl, 1% Triton X-100, 100 mM MES buffer (pH 6.0), 0.6 mM UDP-Gal, 5,000 dpm/l UDP-[ 14 C]Gal (NEN Life Science Products), the enzyme solution, and p-nitrophenyl-␤-D-xylopyranoside (2 mM) in total volume of 50 l. After incubation at 37°C for 30 min, the reaction mixture was applied onto a Sep-Pak C 18 cartridge (Waters), and the product was eluted with 5 ml of methanol. The radioactivity in the eluates was measured in a liquid scintillation counter (Beckman).
Cytostaining-To analyze the subcellular localization of WT and mutated XGalT-1 enzymes, constructs for Myc tag enzyme expression were transiently transfected into pgsB-761 cells. Cells transfected with WT or mutant cDNAs were fixed in 3.7% paraformaldehyde in PBS for 5 min, then permeabilized with PBS containing 0.1% Triton X-100. They were immunostained with mouse anti-Myc tag antibody and FITC-conjugated goat anti-mouse IgG, then observed under MRC-1024 confocal imaging system (Bio-Rad).

Mutations in XGalT-1 Gene in the Patient with Progeroid
Type of E-D Syndrome-The entire coding region of XGalT-1 was amplified by reverse transcription PCR and sequenced. Just two changes of G and T to A and C at 186 and 206, respectively, were detected (Fig. 1). To perform pedigree analysis, we determined the genomic DNA sequences encompassing the A186D and L206P mutations. Consequently, comparison of the genomic and cDNA sequences indicated that two mutations are present in the same exon (Fig. 2). As shown in Fig. 1, the unaffected parents and two siblings were heterozygous for either one of the two different mutations and normal, while the patient had both of two different mutant genes.
Functions of the Products from Mutant cDNAs-To confirm whether the A186D and L206P mutations were responsible for the defects in the synthesis of GAGs, we examined the functions of the expressed mutant cDNAs. XGalT-1-deficient mutant CHO line pgsB-761 (8) was transfected with WT, A186D and L206P cDNAs to examine the restoration of the expression of proteoglycans. A186D could restore fairly well and similarly to WT, while L206P could not at all (Fig. 3A). The enzyme activities in the total cell lysate of these transfectant cells were examined, resulting in the following patterns of activities: A186D showed ϳ50% activity of WT, and L206P was almost null (Fig. 3B, left), despite the expression levels of those products being almost equivalent (Fig. 3C).
Furthermore, soluble enzymes fused to protein A were ex- Pedigree and sequence analysis of genomic DNAs from the patient's family members. DNA segments were amplified from genomic DNAs by PCR as described under "Experimental Procedures." The amplified products were directly sequenced using primer XGTF460. The C 3 A transition changes the sequence of codon 186 from GCC to GAC and resulted in an Ala to Asp substitution. The T 3 C transition changes codon 206 from CTC to CCC, resulting in a Leu to Pro substitution. pressed in L cells and were concentrated 100-fold and served for enzyme assay. Again, A186D showed low activity and L206P showed no activity (Fig. 3B, right). Intracellular Localization of the Mutant Enzymes-Intracellular localization of the mutant enzymes derived from the defined mutant cDNAs was analyzed using Myc-tagged constructs. WT and A186D enzymes showed a similar Golgi pattern. In contrast, L206P was detected in the cytoplasm with a diffuse pattern (Fig. 4). Although the disruption of Golgi targeting of L206P enzyme is an interesting finding, it seems not direct mechanisms for the functional defects as a galactosyltransferase, because the soluble L206P enzyme also completely lost the enzyme activity. Fig. 2, the mutated A186D and L206P lie in the conserved region of the catalytic domain of XGalT-1 protein. In particular, L206 was strictly conserved in all mammalian ␤1,4-galactosyltransferases cloned to date and C. elegans sqv-3 (6). The function of Leu 206 is unknown, but it may be involved in an essential step of the enzyme action. Moreover, replacement by proline, which has been shown to disrupt ␣-helices, would probably result in a significant conformational change, loss of activity, and a change in intracellular trafficking.

Significance of the Mutations on the Molecular Function of XGalT-1-As shown in
Thus, this study has shown that mutations in XGalT-1 gene, which catalyzes the second glycosyl transfer step in the biosynthesis of GAGs, is the primary genetic defect in the progeroid variant of E-D syndrome and that it is inherited in an autosomal recessive manner based on identification of the gene mutations and characterization of the gene products. The incidence of the progeroid variant of E-D syndrome is too low to sufficiently analyze various types of mutations in XGalT-1 gene. However, results reported here clearly indicate the importance of the intact synthesis of GAGs on proteoglycans in the development of the wide variety of tissues. In particular, GAGs seem to be involved in the process of senescence, and the availability of XGalT-1 gene might provide a useful probe to substantially elucidate the molecular mechanisms. XGalT-1 cDNAs. Full-length cDNAs inserted into pCDNA3 with a Myc epitope tag at the 5Ј end of the ORF were transfected into pgsB-761, then expression of heparan sulfate was analyzed on a FACSCalibur (Becton Dickinson) using heparan sulfate-specific mAb HepSS-1. Thin lines are mAb, and thick lines are controls. B, analysis of enzyme activity. The rate of galactose transfer to p-nitrophenyl-␤-D-xylopyranoside (2 mM) was measured using 25 g each of the total cell lysate (left) or protA-XGalT-1 (⌬1-53) purified from 2.5 l of the condition medium (right) as described previously (5). C, Western blot analysis of Myctagged XGalT-1 and mutants expressed in pgsB-761. The transfected cells were lysed, and total cell lysate (50 g) was separated by SDSpolyacrylamide gel electrophoresis and blotted with mouse anti-Myc tag antibody as described under "Experimental Procedures."