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Defective Glycosylation of Decorin and Biglycan J Mol Med DOI 10.1007/s00109-006-0046-4 ORIGINAL ARTICLE Daniela G. Seidler . Muhammad Faiyaz-Ul-Haque . Uwe Hansen . George W. Yip . Syed H. E. Zaidi . Ahmad S. Teebi . Ludwig Kiesel . Martin Götte Defective glycosylation of decorin and biglycan, altered collagen structure, and abnormal phenotype of the skin fibroblasts of an Ehlers–Danlos syndrome patient carrying the novel Arg270Cys substitution in galactosyltransferase I (β4GalT-7) Received: 4 August 2005 / Accepted: 20 January 2006 # Springer-Verlag 2006 Abstract The Ehlers–Danlos syndrome (EDS) is a het- erogeneous group of connective tissue disorders affecting skin and joint function. Molecular defects in extracellular matrix proteins, including collagen (type I, III, and V) and tenascin X are associated with different forms of EDS. Compound heterozygous mutations in the B4GALT7 gene, resulting in aberrant glycosylation of the dermatan sulfate proteoglycan decorin, had been described in a single patient affected with the progeroid form of EDS. We have studied the molecular phenotype of decorin, biglycan, and collagen type I containing fibrils in skin fibroblasts of a patient carrying the novel homozygous C808T point mutation in the B4GALT7 gene, which causes an ARTIN O¨TTE ANIELA EIDLER D. G. Seidler . U. Hansen M G D G. S Department of Physiological Chemistry and Pathobiochemistry, received his Ph.D. in biochem- graduated in 1999 from the Münster University Hospital, istry from the University of University of Heidelberg and Münster, Germany Göttingen, Germany. After started working in the matrix postdoctoral training in Merton biology and decorin field at the ’ M. Faiyaz-Ul-Haque Bernfield s laboratory at Har- Institute of Physiological Department of Pathology & Laboratory Medicine, vard Medical School, he be- Chemistry and Pathobiochem- King Faisal Specialist Hospital & Research Centre, came a faculty member of istry, University of Münster, Riyadh 11211, Saudi Arabia Münster University Hospital, Germany. Currently, she is Germany, where he is currently working at the Department of G. W. Yip heading the research laboratory Pathology, Anatomy, and Cell Department of Anatomy, of the Department of Obstetrics Biology, Thomas Jefferson National University of Singapore, and Gynecology. His main re- University, USA. Singapore 117597, Singapore search interests include the role of proteoglycans and glycos- A. S. Teebi aminoglycans in inflammation, Section of Clinical Genetics and Dysmorphology, angiogenesis, and tumorigenesis. The Hospital for Sick Children, Toronto, Ontario, Canada Arg270Cys substitution in β4GalT-7. Compared to control S. H. E. Zaidi fibroblasts, galactosyltransferase activity in β4GalT- Division of Cardiology, Department of Medicine, 7Arg270Cys cells was approximately three times reduced University Health Network, over a temperature range of 25–41°C. Pulse-chase Toronto, Ontario, Canada experiments and confocal microscopy demonstrated that L. Kiesel . M. Götte (*) synthesis and secretion of decorin were normal in β4GalT- Department of Obstetrics and Gynecology, 7Arg270Cys cells. However, about 50% of decorin were Münster University Hospital, synthesized as a protein core in addition to its proteoglycan 48149 Münster, Germany e-mail: [email protected] form. Biglycan was found in a monoglycanated form in Tel.: +49-251-8356117 addition to its mature form. Glycosaminoglycan chains Fax: +49-251-8355928 were of the dermatan/chondroitin sulfate type both in β4GalT-7Arg270Cys and control cells, and epimerization was sis of mature decorin and biglycan and reduced epimer- reduced for decorin and biglycan. Compared to control ization of the glycosaminoglycan chain of decorin. In cells, β4GalT-7Arg270Cys cells showed altered, highly addition, morphological alterations and an intracellular spread or stretched phenotypes and decreased proliferation accumulation of degradative vacuoles were seen in rates. At the ultrastructural level, an intracellular accumu- β4GalT-7Arg270Cys cells. Furthermore, analysis of the lation of multiple secondary lysosomes and degenerative collagen fibrils showed that the β4GalT-7-deficient cells vacuoles was seen in β4GalT-7Arg270Cys cells. Further- have a different suprastructure, no banded collagen fibrils, more, the collagen suprastructures were altered in the and an altered ratio of α(1) to α(2) chains compared to β4GalT-7Arg270Cys cells. The reduced β4GalT-7 activity control cells. In addition, β4GalT-7Arg270Cys cells exhibited resulting in defective glycosylation of decorin and biglycan reduced cell proliferation rates compared to controls. The may be responsible for the complex molecular pathology in changes in decorin and biglycan glycosylation might be β4GalT-7 deficient EDS patients, given the role of these responsible for several aspects of the EDS phenotype in proteoglycans in bone formation, collagen fibrillogenesis, β4GalT-7-deficient patients, including collagen fibrillo- and skeletal muscle development. genesis and defective wound repair. Keywords Xylosylprotein 4-beta-galactosyltransferase . Ehlers–Danlos syndrome . Dermatan sulfate Materials and methods proteoglycan . Connective tissue diseases . Galactosyltransferases . Glycosaminoglycans Cell culture Skin fibroblasts from an EDS patient carrying the C808T Introduction mutation in the B4GALT7 gene have been recently described [11, 12]. Patient-derived (passages 4–9) and Decorin and biglycan are members of the small leucine-rich age-matched donor-derived skin fibroblasts were cultured proteoglycan family [1, 2], which are important constituents in modified Eagle’s minimum essential medium (MEM) of interstitial extracellular matrices. Both are characterized with Earle’s salts, 10% fetal calf serum (FCS) (Biochrom, by a 45–50 kDa protein core containing 10 tandem leucine- Berlin, Germany), and supplements as described before rich repeat sequences flanked by cysteine-rich disulfide [13]. For the preparation of collagens, cells were cultured loops. Decorin is covalently linked with one and biglycan for 14 days in the presence of 1 mM ascorbate (Sigma- with two glycosaminoglycan chains of the chondroitin/ Aldrich) [13]. dermatan sulfate type, respectively [1]. While decorin is involved in the regulation of cell adhesion and migration, angiogenesis, and growth factor signalling [3], both decorin Cell proliferation assays and biglycan play important roles in bone formation and the regulation of collagen fibrillogenesis [2, 4, 5]. MTT assay Metabolic activity as a readout of cell viability In humans, aberrant glycosaminoglycan substitution of was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyl- decorin was observed in a progeroid syndrome patient [6, 7] tetrazolium bromide (MTT); 2500 fibroblasts per well who carried compound heterozygous mutations in the were seeded in 96-well plates and incubated overnight. B4GALT7 gene encoding galactosyltransferase I (β4GalT- MTT test was carried out as described previously [13]. 7; E.C. 2.4.1.133), which catalyses the transfer of galactose After an overnight incubation at 37°C, the optical density to the xylose residue forming the linkage region of at 595 nm was measured using a microplate reader. proteoglycans [7–10]. This patient’s fibroblasts displayed reduced galactosyltransferase activity [7] and while bio- BrdU assay The colorimetric cell proliferation ELISA kit synthesis of large chondroitin/dermatan sulfate proteogly- (Roche Diagnostics, Penzberg, Germany), which measures cans and heparan sulfate proteoglycans was normal, 20– incorporation of BrdU into newly synthesized DNA, was 80% of the decorin core-protein was substituted with a used exactly as described by the manufacturer to quantify glycosaminoglycan chain [6, 7, 9, 10]. proliferation of fibroblasts. In a recent report, two patients affected with Ehlers– Danlos syndrome (EDS) exhibited a phenotype [11] similar to the previously described patient [6]. The patients showed Metabolic labelling and proteoglycan isolation the typical EDS features including craniofacial appearance and skeletal abnormalities. However, their skin was not Biosynthesis of decorin and biglycan was examined by remarkably loose and no progeroid appearance was seen. metabolic labelling with either [35S]sulfate or [3H]leucine Sequence analysis of the B4GALT7 gene from affected or [35S]sulfate and [35S]methionine by incubation for 12 h patients identified a homozygous C808T substitution as described [14]. After ammonium sulfate precipitation causing an Arg270Cys change in the C-terminal catalytic (70%), conditioned media were ultracentrifuged. The region of β4GalT-7 [11]. In this study, we demonstrate that precipitate was dissolved in 0.1 M TRIS/HCl, pH 7.4, cultured skin fibroblasts from one of these patients exhibit 1 M NaCl, 0.5% Triton X-100, 0.5% Na-deoxycholate, and reduced galactosyltransferase activity, defective biosynthe- proteinase inhibitors. Immunoprecipitation of decorin [14] and biglycan [15] with specific antisera was performed as (BioSepra, Cergy-Saint-Christophe, France) Pasteur pi- described. pette column. After washing with 300 mM NaCl, 20 mM Tris/HCl, pH 7.4, and elution, decorin was immunopre- cipitated as described above. The supernatants, containing Pulse-chase analysis of decorin biosynthesis biglycan, and released decorin were fractionated on a Superose-6 column. The glycosaminoglycan chains of Cells were preincubated in MEM w/o sulfate and methio- decorin and biglycan were subsequently released by β- nine (Gibco) containing 4% FCS dialyzed against the same elimination and
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