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.

Embryonic stem (ES) cells can be cultured in conditions thateither maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe its structural transition from an unusually low-sulfated variant in ES cells to a more highly sulfated form in fluorescence-activated cell sorting-purified neural progenitor cells. The characteristic domain structure of HS was retained during this transformation. However, qualitative variations in surface sulfation patterns between ES and differentiated cells were revealed using HS epitope-specific antibodies and the HS-binding growth factor fibroblast growth factor 2 (FGF-2). Expression profiles of the HS modification enzymes indicated that both "early" (N-sulfotransferases) and "late" (6O-and 3O-sulfotransferases) sulfotransferases contributed to the alterations in sulfation patterning. An HS-null ES line was used to demonstrate the necessity for HS in neural differentiation. HS is a coreceptor for many of the protein effectors implicated in pluripotency and differentiation (e.g., members of the FGF family, bone morphogenic proteins, and fibronectin). We suggest that the stage-specific activities of these proteins are finely regulated by dynamic changes in sulfation motifs in HS chains.

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Virions of lettuce infectious yellows virus (LIYV ; genus Crinivirus) were purified from LIYV-infected plants and their protein composition was analysed by SDS-PAGE and immunoblotting. Virion preparations contained the major capsid protein (CP), but the minor capsid protein (CPm), p59 and the HSP70 homologue were also identified by immunoblot analysis. Immunogold labelling analysis showed that CP constituted the majority of the LIYV virion capsid, but CPm was also part of the capsid and localized to one end of the virion, similar to the polar morphology seen for viruses in the genus Closterovirus. p59 and the HSP70 homologue were not detected on virions by immunogold labelling, but were always detected in virion preparations by immunoblot analysis. Purified LIYV virions were used for in vitro acquisition analysis with Bemisia tabaci whiteflies and were efficiently transmitted to plants. Infectivity neutralization analyses were done using antisera to the LIYV-encoded CP, CPm, p59 and HSP70 homologue. Only antiserum to the CPm effectively neutralized LIYV transmission by B. tabaci. These data suggest that the LIYV-B. tabaci transmission determinants are associated with purified virions, and that the LIYV virion structural protein CPm is involved in transmission by B. tabaci.