Structural characterization and antithrombin activity of dermatan sulfate purified from marine clam Scapharca...

Glycosaminoglycans from the body of marine clam Scapharca inaequivalvis were extracted at about 0.15- 0.18 mg/g of dry tissue, composed of dermatan sulfate (DS) (approx. 74%) and heparan sulfate (26%). After treatment with nitrous acid, DS was isolated for further complete structural characterization. Agarose-gel electrophoresis in combination with various enzymes, chondroitin ABC lyase, chondroitin B lyase, chondroitin ACII lyase from Arthrobacter aurescens, and chondroitin AC lyase from Flavobacterium heparinum, confirmed the DS nature of this polysaccharide. Furthermore, by evaluating the unsaturated disaccharides produced by the action of the various lyases, this natural polymer was found to be composed of approx. 75% of disaccharides containing iduronic acid (IdoA) mainly found in disaccharides monosulfated in position 4 of N-acetylgalactosamine (GalNAc) and disulfated in position 2 of the IdoA and 4 of GalNAc (disaccharide B typical of DS). In contrast, glucuronic acid was found to be mainly associated with the nonsulfated disaccharide (approx. 92%), while the rest formed low percentages of monosulfated disaccharides in position 4 or 6 of GalNAc preferentially located inside the chains. Generally, this GAG possesses a peculiar structure, due to the presence of significant amounts of nonsulfated disaccharide mainly located close to the nonreducing end, to the elevated percentage of the disaccharide B, and to the presence of not previously reported low amounts of the disaccharide monosulfated in position 2 of the uronic acid. S. inaequivalvis DS was also found to have a mean molecular mass of approx. 27,000 Da and a mean charge density of 1.10 that increases to 1.54 for the carbohydrate backbone composed of IdoA residues. (1)H-NMR and (13)C-NMR analyses confirmed the nature of S. inaequivalvis polymer revealed by the presence of signals related to DS corresponding to the residue of IdoA and GalNAc mainly sulfated at the C4 along with the presence of a signal belonging to the residue of H1 IdoA-2SO(4). S. inaequivalvis DS was further depolymerized by partial controlled digestion with chondroitinase ABC and separated into oligosaccharides by online HPLC/ESI-MS to obtain sequence information. The most prominent generated oligosaccharides comprised the repeating unit Delta Hex-GalNAcSO(4) thus confirming the results obtained by disaccharide analysis and the structures of the major oligosaccharides (from 6- to 10-mer) confirmed, by means of the LC-MS, the presence of approx. 20% of nonsulfated disaccharide. Furthermore, a minor but significant percentage of a monosaccharide having an m/z 300 and corresponding to GalNAcSO(4) belonging to the DS nonreducing end was observed along with saturated hexasaccharide derived from the nonreducing terminus of the intact DS ending with a uronic acid residue. Finally, S. inaequivalvis DS was calculated to possess a high heparin cofactor II activity of 169.2 +/- 10.7% fairly similar to that of several DS samples purified from porcine and bovine tissues.