MAB570 Sigma-AldrichAnti-Heparin Antibody, clone A7.10

Recently heparan sulfate was proposed as the host cell receptor for the dependovirus, adeno-associated virus type 2 (AAV2). We show that although heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with AAV2 binding to cells or recombinant AAV2 transduction efficiency. Experiments with either mutant CHO cells or cells treated with chlorate to remove sulfate groups showed that sulfation was not absolutely required for infection or binding: in the absence of cell surface sulfation, recombinant AAV2 was still able to be transduced in previously permissive cells. Heparin is commonly used as a substitute in studies of the interaction between heparan sulfate and ligand, and we demonstrate that the binding affinity of AAV2/heparin is low, with a K(d) value of approximately 2.0 nM. A study of the direct interaction between AAV2 and artificial glycosaminoglycans showed that a high degree of sulfation on heparin was critical for the ability to bind AAV2 and compete rAAV2 transduction and that both O- and N-sulfate groups are required. Overall, our data suggest that, as has been shown for other viruses, the presence of a high-affinity AAV2 receptor mediates AAV2 infection in addition to the low-affinity heparan sulfate binding.

Toxoplasma gondii is one of the most widespread parasites of humans and animals. The parasite has a remarkable ability to invade a broad range of cells within its mammalian hosts by mechanisms that are poorly understood at the molecular level. This broad host cell specificity suggests that adhesion should involve the recognition of ubiquitous surface-exposed host molecules or, alternatively, the presence of various parasite attachment molecules able to recognize different host cell receptors. We have discovered a sugar-binding activity (lectin) in tachyzoites of T. gondii that plays a role in vitro in erythrocyte agglutination and infection of human fibroblasts and epithelial cells. The ability to agglutinate erythrocytes can be reversed by a variety of soluble glycoconjugates, of which heparin, fucoidan, and dextran sulfate were the most effective. Interestingly, infectivity of tachyzoites for human foreskin fibroblasts, cells that are commonly used to grow T. gondii in vitro, was increased by low concentrations of the sulfated glycoconjugates that inhibited hemagglutination activity (i.e. dextran sulfate and fucoidan) whereas high concentrations inhibited parasite infection. Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation on the host cells reduced Toxoplasma infectivity. Finally, Toxoplasma tachyzoites showed a reduced ability to infect epithelial cell mutants deficient in the biosynthesis of surface proteoglycans. The probable identity of the hemagglutinin(s) was investigated by 1) direct binding of red blood cells to filter blots of Toxoplasma proteins separated by polyacrylamide gel electrophoresis, and 2) binding of metabolically labeled parasite proteins to fixed mammalian cells. Three parasite bands were thus identified as candidate adhesins. These results suggest that attachment of T. gondii to its target cell is mediated by parasite lectins and that sulfated sugars on the surface of host cells may function as a key parasite receptor.

Heparin and heparan sulfate are related glycosaminoglycans which demonstrate high-affinity interactions with a number of proteins, including antithrombin III. The immunogenicity of heparin has been reported previously employing heparin-protein conjugates as immunogens and as antigens in solid-phase assays. Previous studies also demonstrate that anti-heparin antibodies play a role in autoimmune diseases including systemic lupus and anti-phospholipid syndrome and in patients who receive heparin for therapeutic purposes. In the current study, we investigated the expression of monoclonal anti-heparin antibodies in nonimmunized, autoimmune MRL/lpr/lpr++ mice employing a liquid-phase radioimmunoassay. The Kd of monoclonal IgG2b autoantibodies for heparin was approximately 10(-8)M. Anti-heparin antibodies were precipitating, and were not polyreactive. The IgG monoclonal antibodies described in this study represent an immunological instance of a specific, high-affinity heparin-protein interaction.