Tunicate Heparan Sulfate Enriched in 2-Sulfated β-Glucuronic Acid: Structure, Anticoagulant Activity, and Inhibitory Effect on the Binding of Human Colon Adenocarcinoma Cells to Immobilized P-Selectin

Abstract

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and β-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated β-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

Non-Anticoagulant Heparan Sulfate from the Ascidian Phallusia nigra Prevents Colon Carcinoma Metastasis in Mice by Disrupting Platelet-Tumor Cell Interaction

“…Despite the intrinsic heterogeneity, the distinct disaccharide components of mammalian HSs are often arranged as repetitive building-blocks along their chains [26]. Previous two-dimensional (2D) NMR spectra assessments performed by our group have already demonstrated that PNH possesses increased proportions of disaccharides containing N-acetyl α-glucosamine and/or β-glucuronic acid (>50%), which is characteristic of HSs, but they are randomly arranged along their chains [21].…”

“…Fractions containing each peak were pooled together, dialyzed against distilled water, lyophilized and stored at −20 • C for further utilization. PNH disaccharide composition was previously shown by NMR analysis [21].…”

“…Once we have confirmed the purity and physical-chemical features of the PNH molecule [21], we began to evaluate the in vitro antitumor activity of this molecule. First, we assessed whether PNH has cytotoxic effects on the MC-38 colon carcinoma cell line using an MTT assay.…”

“…Adult specimens of the ascidian P. nigra were collected in the Ilha Grande Bay (Rio de Janeiro state, Brazil) by scuba diving. Sulfated polysaccharides from the viscera of P. nigra were extracted through proteolytic digestion with papain, and then, PNH was purified with anion-exchange chromatography, as previously described [21,43]. Briefly, crude polysaccharide extracts from P. nigra were applied into a DEAE-cellulose column, equilibrated with 50 mM sodium acetate (pH 5.0), and then eluted through a linear gradient of 0.5→1.0 M NaCl.…”

“…Additionally, platelet and tumor cell interaction is reinforced by fibrin accumulation around these cloaks, originating a circulating tumor microemboli (CTM) [3]. [21]. (B) Proportions of PNH major disaccharides components.…”