Stéphane Sarrazin

Heparan Sulfate Proteoglycans

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein-heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level.

Heparan Sulfate Mimicry A SYNTHETIC GLYCOCONJUGATE THAT RECOGNIZES THE HEPARIN BINDING DOMAIN OF INTERFERON-γ INHIBITS THE CYTOKINE ACTIVITY

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-γ (IFNγ), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNγ monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNγ. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Å-long spacer termed 2O10, displays high affinity for the cytokine and inhibits IFNγ-HS binding with an IC50 of 35–40 nm. Interestingly, this molecule also inhibits the binding of IFNγ to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNγ activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNγ recognition, providing a new strategy to inhibit IFNγ in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create”tailor-made“sequences based on the HS template to isolate therapeutic activities.

Secondary Storage of Dermatan Sulfate in Sanfilippo Disease

Mucopolysaccharidoses are a group of genetically inherited disorders that result from the defective activity of lysosomal enzymes involved in glycosaminoglycan catabolism, causing their intralysosomal accumulation. Sanfilippo disease describes a subset of mucopolysaccharidoses resulting from defects in heparan sulfate catabolism. Sanfilippo disorders cause severe neuropathology in affected children. The reason for such extensive central nervous system dysfunction is unresolved, but it may be associated with the secondary accumulation of metabolites such as gangliosides. In this article, we describe the accumulation of dermatan sulfate as a novel secondary metabolite in Sanfilippo. Based on chondroitinase ABC digestion, chondroitin/dermatan sulfate levels in fibroblasts from Sanfilippo patients were elevated 2–5-fold above wild-type dermal fibroblasts. Lysosomal turnover of chondroitin/dermatan sulfate in these cell lines was significantly impaired but could be normalized by reducing heparan sulfate storage using enzyme replacement therapy. Examination of chondroitin/dermatan sulfate catabolic enzymes showed that heparan sulfate and heparin can inhibit iduronate 2-sulfatase. Analysis of the chondroitin/dermatan sulfate fraction by chondroitinase ACII digestion showed dermatan sulfate storage, consistent with inhibition of iduronate 2-sulfatase. The discovery of a novel storage metabolite in Sanfilippo patients may have important implications for diagnosis and understanding disease pathology.

Guanidinylated Neomycin Mediates Heparan Sulfate-dependent Transport of Active Enzymes to Lysosomes

Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, beta-D-glucuronidase (GUS) and alpha-L-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells.

Insights into the Mechanism by Which Interferon-γ Basic Amino Acid Clusters Mediate Protein Binding to Heparan Sulfate

The extensive functional repertoire of heparin and heparan sulfate, which relies on their ability to interact with a large number of proteins, has recently emerged. To understand the forces that drive such interactions the binding of heparin to interferon-γ (IFNγ), used as a model system, was investigated. NMR-based titration experiments demonstrated the involvement of two adjacent cationic domains (D1: KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal sequence of the cytokine. Kinetic analysis showed that these two domains contribute differently to the interaction: D1 is required to form a complex and constitutes the actual binding site, whereas D2, although unable to associate with heparin by itself, increased the association rate of the binding. These data are consistent with the view that D2, through nonspecific electrostatic forces, places the two molecules in favorable orientations for productive binding within the encounter complex. This mechanism was supported by electrostatic potential analysis and thermodynamic investigations. They showed that D1 association to heparin is driven by both favorable enthalpic and entropic contributions, as expected for a binding sequence, but that D2 gives rise to entropic penalty, which opposes binding in a thermodynamic sense. The binding mechanism described herein, by which the D2 domain kinetically drives the interaction, has important functional consequences and gives a structural framework to better understand how specific are the interactions between proteins and heparin.

Cooperative, Heparan Sulfate-Dependent Cellular Uptake of Dimeric Guanidinoglycosides

Oligoarginine and guanidinium‐rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell‐surface heparan sulfate proteoglycans as part of their cell‐entry pathway. Unlike for other guanidinium‐rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell‐surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell‐surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild‐type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin–phycoerythrin‐Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell‐surface receptors. Competition studies with chemically desulfated or carboxy‐reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell‐surface heparan sulfate proteoglycans.

A Genetic Model of Substrate Reduction Therapy for Mucopolysaccharidosis

Background: Treatment of neuropathology in mucopolysaccharidoses may be possible by substrate reduction therapy. Results: A genetic model of substrate reduction therapy for mucopolysaccharidosis type IIIa ameliorates disease pathology in the brain. Conclusion: Partial inhibition of glycosaminoglycan biosynthesis may be useful for treating mucopolysaccharidoses type III. Significance: Proof of principle is presented that inhibition of heparan sulfate synthesis might prove beneficial for treating mucopolysaccharidoses. Inherited defects in the ability to catabolize glycosaminoglycans result in lysosomal storage disorders known as mucopolysaccharidoses (MPS), causing severe pathology, particularly in the brain. Enzyme replacement therapy has been used to treat mucopolysaccharidoses; however, neuropathology has remained refractory to this approach. To test directly whether substrate reduction might be feasible for treating MPS disease, we developed a genetic model for substrate reduction therapy by crossing MPS IIIa mice with animals partially deficient in heparan sulfate biosynthesis due to heterozygosity in Ext1 and Ext2, genes that encode the copolymerase required for heparan sulfate chain assembly. Reduction of heparan sulfate by 30–50% using this genetic strategy ameliorated the amount of disease-specific biomarker and pathology in multiple tissues, including the brain. In addition, we were able to demonstrate that substrate reduction therapy can improve the efficacy of enzyme replacement therapy in cell culture and in mice. These results provide proof of principle that targeted inhibition of heparan sulfate biosynthetic enzymes together with enzyme replacement might prove beneficial for treating mucopolysaccharidoses.