Multiple interactions of HIV-I tat protein with size-defined heparin oligosaccharides

Tat protein, a transactivating factor of the human immunodeficiency virus t ype I, acts also as an extracellular molecule. Heparin affects the bioavail ability and biological activity of extracellular Tat (Rusnati, Prl., Coltri ni, D., Oreste, P., Zoppetti, G., Albini, k, Noonan, D., D'Adda di Fagagna, F., Giacca, M., and Presta, M. (1997) J. Biol. Chem. 272, 11313-11320). He re, a series of homogeneously sized, H-3-labeled heparin fragments were eva luated for their capacity to hind to free glutathione S-transferase (GST)-T at protein and to immobilized GST-Tat. Hexasaccharides represent the minimu m sized heparin fragments able to interact with GST-Tat at physiological io nic strength. Also, the affinity of binding increases with increasing the m olecular size of the oligosaccharides, with large fragments (greater than o r equal to 18 saccharides) approaching the affinity of full-size heparin. 6 -Mer heparin binds GST-Tat with a dissociation constant (K-d) equal to 0.7 +/- 0.4 mu M and a molar oligosaccharide:GST-Tat ratio of about 1:1. Intera ction of GST-Tat with 22-mer or full-size heparin is consistent instead wit h two-component binding. At subsaturating concentrations, a single molecule of heparin interacts with 4-6 molecules of GST-Tat with high affinity K-d values in the nanomolar range of concentration at saturating concentrations , heparin binds GST-Tat with lower affinity (K-d, values in the micromolar range of concentration) and a molar oligosaccharide:GST-Tat ratio of about 1:1. In agreement with the binding data, a positive correlation exists betw een the size of heparin oligosaccharides and their capacity to inhibit cell internalization, long terminal repeat-transactivating activity of extracel lular Tat in HL3T1 cells, and its mitogenic activity in murine adenocarcino ma T53 Tat-less cells. The data demonstrate that the modality of heparin-Tat interaction is strong ly affected by the size of the saccharide chain. The possibility of establi shing multiple interactions increases the affinity of large heparin fragmen ts for Tat protein and the capacity of the glycosaminoglycan to modulate th e biological activity of extracellular Tat.