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

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.

Demystifying a hexuronic acid ligand that recognizes Toxoplasma gondii and blocks its invasion into host cells

Toxoplasma gondii is a ubiquitous eukaryotic pathogen responsible for toxoplasmosis in humans and animals.

Disrupted Synthesis of a Di-N-acetylated Sugar Perturbs Mature Glycoform Structure and Microheterogeneity in theO-Linked Protein Glycosylation System ofNeisseria elongatasubsp.glycolytica

ABSTRACTThe genusNeisseriaincludes three major species of importance to human health and disease (Neisseria gonorrhoeae,Neisseria meningitidis, andNeisseria lactamica) that express broad-spectrumO-linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain ofNeisseria elongatasubsp.glycolytica, a unique tetrasaccharide glycoform consisting of di-N-acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di-N-acetyl hexuronic acid and aN-acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di-N-acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in theN. elongatasubsp.glycolyticagenome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di-N-acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms.IMPORTANCEBroad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants ofN. elongatasubsp.glycolyticathat define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di-N-acetyl-glucuronic acid and singular properties related to the relaxed specificities of theN. elongatasubsp.glycolyticasystem