Caracterização estrutural e avaliação da atividade anticoagulante de condroitim sulfato de lula Doryteuthis (Loligo) plei

Abstract

Glycosaminoglycans (GAGs) are linear heteropolysaccharides attached to a core protein of proteoglycans (PGs). Chondroitin sulfate (CS) is sulfated GAG composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-galactosamine, this compound has biological activities associated to their interaction with different components of the extracellular matrix. These activities have been related to their structural characteristics due to the high content of sulfate and carboxyl groups originating structural domains which are known to participate in specific physiological functions. This aim of this study was the identification and structural characterization of glycosaminoglycans extracted from squid different tissues (Doryteuthis plei) as well as evaluation of the anticoagulant activity of these compounds. Initially, we determined the procedures GAGs extraction in mantle tissue, fin, tentacles and skin using degradation with proteolytic enzymes and purification using ion exchange chromatography. We carried out the identification of GAG to degradation with specific lyases (chases AC and B from F. heparinum), wherein we evidenced that CS is the principal GAG of the D. plei tissues. Molecular weight of CS had a range of 30-50 kDa. Quantification of sulfate groups demonstrated that these CS had higher ratio of sulfate/hexosamine (1.2), a indicative that these macromolecules were oversulfated. This fact was confirmed by FACE analysis of CS from mantle and fin after digestion with chase AC from A. aurescens where most sulfation was related to the presence of high proportion of di-sulfated residues, ΔDi4,6S (≈55%). Raman spectroscopic and NMR techniques confirmed the increased substitution and sulfation in positions 4 and 6 of GalNAc, respectively. CS of the mantle and fin demonstrated anticoagulant activity associated with inhibition of the intrinsic coagulation pathway by inhibition of factors Xa and IIa. Thus, we conclude that CS-E is the kind of glycosaminoglycan constituent in squid tissues and it has tissue-specific characteristics, playing their anticoagulant activity according to the sulfation pattern.