Malaria infection is initiated when Anopheles mosquitoes inject Plasmodium sporozoites into the skin. Sporozoites subsequently reach the liver, invading and developing within hepatocytes. Sporozoites contact and traverse many cell types as they migrate from skin to liver; however, the mechanism by which they switch from a migratory mode to an invasive mode is unclear. Here, we show that sporozoites of the rodent malaria parasite Plasmodium berghei use the sulfation level of host heparan sulfate proteoglycans (HSPGs) to navigate within the mammalian host. Sporozoites migrate through cells expressing low-sulfated HSPGs, such as those in skin and endothelium, while highly sulfated HSPGs of hepatocytes activate sporozoites for invasion. A calcium-dependent protein kinase is critical for the switch to an invasive phenotype, a process accompanied by proteolytic cleavage of the sporozoite's major surface protein. These findings explain how sporozoites retain their infectivity for an organ that is far from their site of entry.
Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by Type I Herpes Simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.
To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes ( 12 C 6 and 13 C 6 ). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.Metazoans make several types of sulfated glycosaminoglycans (GAGs), 2 including keratan sulfate (KS), chondroitin sulfate/dermatan sulfate (CS/DS), and heparan sulfate/heparin (HS). Each type of chain consists of unique disaccharide units. KS consists of galactose (Gal) and GlcNAc ([Gal1, 4GlcNAc1,3] n ) with variable sulfation at C6 of either sugar. CS/DS assembles as a copolymer of GlcA1,3GalNAc1,4 and then undergoes various processing reactions, including C5 epimerization of a portion of GlcA units to iduronic acid in DS, O-sulfation at C2 and more rarely at C3 of the uronic acids, and O-sulfation at C4 and C6 of the GalNAc residues (1). HS is the most highly modified GAG, consisting initially of GlcA1,4GlcNAc␣1,4 units, which then undergo variable processing by GlcNAc N-deacetylation and N-sulfation, C5 epimerization of some GlcA units to iduronic acid, and O-sulfate addition to C2 of the uronic acids and C6 and more rarely at C3 of the glucosamine units (2). The arrangement of the modified residues along the chain creates binding sites for numerous growth factors, enzymes, and extracellular matrix proteins. The structural variation that can occur makes sulfated GAG chains one of the most complex classes of macromolecules found in nature.GAG fine structure is typically assessed by analyzing the disaccharide composition of an isolated mixture of chains. A number of techniques have been developed to accomplish this task that rely on chemical or enzymatic depolymerization of the chains into their constituent disaccharides, followed by separation via anion exchange chromatography, reversed-phase chromatography with ion pairing agents, or capillary electrophoresis. These techniques separate...
In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2-and VEGF 165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan-protein interactions.antithrombin ͉ glycosaminoglycans ͉ growth factors ͉ viral infection ͉ angiogenesis
A significant need exists for improved biomarkers for differential diagnosis, prognosis and monitoring of therapeutic interventions for mucopolysaccharidoses (MPS), inherited metabolic disorders that involve lysosomal storage of glycosaminoglycans. Here, we report a simple reliable method based on the detection of abundant non-reducing ends of the glycosaminoglycans that accumulate in cells, blood, and urine of MPS patients. In this method, glycosaminoglycans were enzymatically depolymerized releasing unique mono-, di-, or trisaccharides from the non-reducing ends of the chains. The composition of the released mono- and oligosaccharides depends on the nature of the lysosomal enzyme deficiency, and therefore they serve as diagnostic biomarkers. Analysis by liquid chromatography/mass spectrometry allowed qualitative and quantitative assessment of the biomarkers in biological samples. We provide a simple conceptual scheme for diagnosing MPS in uncharacterized samples and a method to monitor efficacy of enzyme replacement therapy or other forms of treatment.
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