Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin Costas C. Chatziioannidis, Nikos K. Karamanos, Stavros T. Anagnostides, Theodore Tsegenidis* Section of Organic Chemistry, Biochemistry and Natural Products, Department of Chemistry, University of Patras, 26500 Patras, Greece (Received 21 May 1998; accepted 4 March 1999) Abstract — A minor low-sulphated dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass (M r ) ranging from 70 to 120 kDa is composed of about two dermatan sulphate chains (M r 33 kDa) bound on a protein core of M r 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the dermatan sulphate chain. © Société française de biochimie et biologie moléculaire / Elsevier, Paris proteoglycans / dermatan sulphate / ray skin 1. Introduction Dermatan sulphate-containing proteoglycans are widely distributed in the extracellular matrix of skin and a variety of other connective tissues [1–4]. The predominant proteoglycan (PG) found in vertebrate skin is decorin, which consists of a protein core of 329 amino acids [5] carrying a single dermatan sulphate (DS) chain [6] and three N-linked oligosaccharides [7]. A small dermatan sulphate-proteoglycan (biglycan), which carries two der- matan sulphate chains and two N-linked oligosaccharides on a core of the same size as decorin’s, was isolated from cultured fibroblasts [8] and from most adult fibrous con- nective tissues [9]. Skin also contains a large chondroitin sulphate-proteoglycan (versican) in small amounts [10, 11]. Studies on chondrichthyes, which are primitive ver- tebrate organisms, have established that shark skin con- tains hyaluronic acid (HA), DS, over-sulphated DS and chondroitin sulphate (CS) [12], chimaera skin contains HA and CS [13], while ray skin showed the presence of DS, very low-sulphated DS (LSDS) and trace amounts of HA [14]. A major proteoglycan, DSI-PG, was isolated from ray skin recently, bearing one dermatan sulphate chain with slightly increased degree of sulphation and oligosaccha- rides containing uronic acid (Chatziioannidis et al., sub- mitted). DSI-PG was almost completely extracted from the tissue by 4 M guanidine hydrochloride (GdnHCl), containing 1.5% Triton X-100 and separated from other possible minor proteoglycans by ion-exchange chroma- tography. DS chains contain significant amounts of disul- phated disaccharides (Δdi-diS) with high variability in the position of sulphation and of neutral monosaccharides. High variability in sulphate distribution [15–17] as well as in the presence of neutral sugar branches [15, 18–23] has also been found in some glycosaminoglycans (GAGs) present in invertebrates. In addition, DSI-PG showed a variation on the protein core size. It was, further, identified that the protein core does not have any similarity with those of small leucine-rich repeat proteoglycans. In the present work the extraction from ray skin (Raja clavata), purification and characterisation of a minor small proteoglycan bearing low-sulphated dermatan sulphate chains is reported. It is also demonstrated that this PG does not interact with hyaluronic acid and does not form self-aggregates. * Correspondence and reprints Abbreviations: HPLC, high-performance liquid chromatogra- phy; PGs, proteoglycans; GAGs, glycosaminoglycans; CS, chondroitin sulphate; DS, dermatan sulphate; HA, hyaluronic acid; LSDS, very low-sulphated dermatan sulphate; Δdi-diS, Δ-disaccharides containing two sulphate groups; GdnHCl, gua- nidine hydrochloride; SDS, sodium dodecyl sulphate; PMSF, phenylmethanesulphonyl fluoride; NEM, N-ethyl maleimide. Biochimie 81 (1999), 187-196 © Société française de biochimie et biologie moléculaire / Elsevier, Paris