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A Genetic Defect in the Biosynthesis of Dermatan Sulfate Proteoglycan

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A Genetic Defect in the Biosynthesis of Dermatan Sulfate Proteoglycan Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1342-1346, February 1990 Medical Sciences A genetic defect in the biosynthesis of dermatan sulfate proteoglycan: Galactosyltransferase I deficiency in fibroblasts from a patient with a progeroid syndrome (galactosyltransferase iI/heparan sulfate/p-nitrophenyl (8-D-xyloside) EDELGARD QUENTIN*, ACHIM GLADEN*, LENNART RODENt, AND HANS KRESSE* *Institute of Physiological Chemistry and Pathobiochemistry, University of Munster, Munster, Federal Republic of Germany; and tDepartments of Medicine and Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294 Communicated by Elizabeth F. Neufeld, November 28, 1989 ABSTRACT A small proteoglycan that contains only a at most 80% and in some experiments as little as 20% into single dermatan sulfate chain is the main proteoglycan synthe- mature proteoglycan molecules. The remainder was secreted sized by skin fibroblasts. Fibroblasts from a patient with in a glycosaminoglycan-free form. progeroidal appearance and symptoms of the Ehlers-Danlos We had discussed previously that the patient could carry a syndrome have a reduced ability of converting the core protein mutant allele yielding a core protein with an absent or buried of this proteoglycan into a mature glycosaminoglycan chain- recognition site for the attachment of the glycosaminoglycan bearing species. This abnormality is the consequence of a chain. In light of the limited induction of glycosaminoglycan deficiency in galactosyltransferase I (xylosylprotein 4- biosynthesis in the presence of low concentrations of p- ,B-galactosyltransferase; EC 2.4.1.133), which catalyzes the nitrophenyl ,B-D-xyloside, which serves as an artificial stimu- second glycosyl transfer reaction in the assembly of the der- lator ofglycosaminoglycan synthesis, an abnormality in one of matan sulfate chain. The glycosaminoglycan-free core protein the enzymes involved in the synthesis of the polysaccharide- secreted by the patient's fibroblasts bears an unsubstituted protein linkage region, GlcA(J31-3)Gal(J31-3)Gal(J31-4)Xyl, was xylose residue. The mutant enzyme is abnormally thermo- also considered. The results presented in this paper provide labile. Preincubation of fibroblasts at 41°C leads to a further evidence that the second explanation is the correct one. reduction in the production ofmature proteoglycan and affects Fibroblasts from the patient contain reduced activities of an the capacity for glycosaminoglycan synthesis on p-nitrophenyl abnormally thermolabile galactosyltransferase I (xylosylpro- f8-D-xyloside more strongly in the mutant than in control cells. tein 4-,B-galactosyltransferase; EC 2.4.1.133), which catalyzes the second glycosyl transfer reaction in the assembly of the The occurrence of inborn errors in the metabolism of con- xylose/serine-linked proteoglycans (see ref. 13 for a review). nective tissue proteoglycans in humans has long been rec- ognized, and over the past two decades the basic genetic MATERIALS AND METHODS defects in many ofthese disorders have been elucidated. The majority of the diseases are caused by faulty degradation of Materials. O-,8-D-Xylopyranosyl-L-serine and O-,/-D-ga- the polysaccharide components ofthe proteoglycans (see ref. lactopyranosyl-(1-4)-O-/3-D-xylopyranosyl-L-serine were 1 for a review). In a few instances, defects in the biosynthesis synthesized as described (14). UDP[4,5-3H]galactose (spe- of proteoglycans have been demonstrated in experimental cific radioactivity, 1.5 GBq/mol) was obtained from DuPont. animals, such as impaired posttranslational processing of the Sodium boro[3H]hydride (929 TBq/mol), L-[4,5-3H]leucine cartilage proteoglycan core protein in nanomelic chicken (2) (1.8 TBq/mol), and sodium [35S]sulfate (carrier-free) were and a tissue-specific deficiency in the biosynthesis of 3'- from Amersham. A high-performance carbohydrate analysis phosphoadenylylsulfate in brachymorphic mice (3). Defec- column was purchased from Millipore Waters; Aminex HPX- tive biosynthesis of proteoglycans is probably the cause of 87 H and TSK DEAE-5PW Bio-Gel columns were from the connective tissue abnormalities observed in several dis- Bio-Rad. eases in humans, including macular corneal dystrophy (4) and Assay of Galactosyltransferases I and II. Human skin fibro- a form of spondyloepiphyseal dysplasia (5), though the de- blasts were cultured as described (15). To minimize the fects in these diseases have not yet been elucidated. concentration of endogenous galactose acceptors, confluent We have previously described a patient who represented a cultures were incubated for 3 hr with 20 ,uM cycloheximide progeroid variant with signs of the Ehlers-Danlos syndrome prior to harvesting according to Esko et al. (16). During the (6). In addition to aged appearance, developmental delay, course of the experiments, however, it became apparent that dwarfism, craniofacial dysproportion, and generalized os- this pretreatment had been unnecessary. Cells were homog- teopenia, this patient suffered from defective wound healing, enized by ultrasonication in 50 mM 2-(N-morpholino)ethane- hypermobilejoints, hypotonic muscles, and loose but elastic sulfonic acid/200 mM KCI/0.05% Triton X-100/20 ,M phen- skin. Biochemically, his cultured skin fibroblasts were de- ylmethylsulfonyl fluoride/2 ,M leupeptin, pH 5.5. Fifty fective in the biosynthesis of a ubiquitous proteoglycan microliters of the suspension containing 50-100 ,g of cell named small dermatan sulfate proteoglycan II (DS-PG II; protein (17) was mixed with 30 ,ul of 16.25 mM Tris/acetate refs. 7 and 8) or decorin (9), which consists of an Mr 36,319 buffer (pH 7.5) containing 37 kBq (86 pmol) of UDP- core protein (10), a single glycosaminoglycan chain on the [3H]galactose, 16.25 mM KCI, 12.5 mM 2,3-dimercaptopro- serine residue at position 4 (11), and either two or three panol, 31 mM MnC12, 0.25 mM ATP, 52 mM CDP-choline, asparagine-bound oligosaccharides (12). The fibroblasts syn- and either 13 mM xylosylserine (galactosyltransferase I) or thesized normal amounts of this core protein but converted 21.8 mM galactosylxylosylserine (galactosyltransferase II, EC 2.4.1.134). After 1 hr at 37°C, the incubation mixture was processed exactly as described (14). Briefly, proteins were The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: DS-PG I1, small dermatan sulfate proteoglycan II. 1342 Downloaded by guest on September 29, 2021 Medical Sciences: Quentin et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1343 precipitated with ethanol, and the soluble material was sub- preincubated for 48 hr at 37°C or 41°C, respectively, and jected to cation-exchange chromatography for the purifica- incubated with [35S]sulfate in the presence of p-nitrophenyl tion ofthe radioactive product. Without exception, the purity ,-D-xyloside for 4 hr at 37°C. Proteoglycans were removed of the radioactive products, Gal-Xyl-Ser or Gal-Gal-Xyl-Ser, by an (NH4)2SO4 precipitation step, and induced chains were was subsequently checked by high-voltage electrophoresis purified by chromatography on Dowex AG 1X2 (200-400 (14) and determination of the 3H radioactivity in 1-cm paper mesh). For the determination ofthe glycosaminoglycan chain segments. By-products accounted for up to 10% in the length, DS-PG II was isolated after an incubation period of 4 control cell lines and 50o in the patient's fibroblasts, and the hr in the presence of 0.37 MBq of [35S]sulfate per ml. ion-exchange data were corrected correspondingly. Dermatan sulfate chains were obtained by a 8-elimination Preparation of DS-PG II and Its Core Protein. Conditioned reaction and chromatographed on a calibrated Sephacryl medium that contained only insulin and transferrin as exog- S-300 column as described (18). enously supplied proteins (18) served as the source of unla- beled DS-PG II and core protein. Glycosaminoglycan-free core protein and DS-PG II were separated from each other by RESULTS anion-exchange chromatography (12). Core protein-con- Presence of Unsubstituted Xylose Residues. Intact DS-PG II taining fractions were dialyzed against 0.1% Triton X-100, and its glycosaminoglycan-free core protein from the secre- lyophilized, and immune precipitated with an affinity- tions of the patient's fibroblasts were separately purified for purified antiserum against DS-PG II core protein (12). DS-PG an analysis of constituents of the polysaccharide protein II-containing fractions were similarly dialyzed and Iyo- linkage region. Alkaline borotritide reduction of both prep- philized but were then exhaustively digested with chondroitin arations led to the incorporation of similar amounts of ABC Iyase (19). Both preparations were subjected to prepar- radioactivity into glycosaminoglycan-free core protein and ative SDS/polyacrylamide gel electrophoresis (20). Proteins chondroitin ABC lyase-digested DS-PG II, the former con- were visualized by treating the gels with 1 M KCI (21), and taining 77% of the radioactivity of the latter. After further bands ofinterest were electroeluted. After drying the samples purification by descending paper chromatography, material under reduced pressure, salts were removed by washing with behaving as authentic [1-3H]xylitol was found upon chroma- methanol. The secretion of [3H]leucine-labeled DS-PG II and tography on either an Aminex HPX-87 H or a carbohydrate core protein was followed as described (12). analysis
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