Rusnati M, Coltrini D, Oreste P, Zoppetti G, Albini A, Noonan D, d'Adda di Fagagna F, Giacca M, Presta M
Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 25123 Brescia, Italy.
J Biol Chem. 1997 Apr 25;272(17):11313-20. doi: 10.1074/jbc.272.17.11313.
Human immunodeficiency virus type 1 (HIV-1) Tat protein is released from infected cells. Extracellular Tat enters the cell where it stimulates the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Benelli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialunga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) and transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10, 1733-1739) activity of extracellular Tat. Here we demonstrate that heparin binds specifically to recombinant HIV-1 Tat produced as glutathione S-transferase (GST) fusion protein and immobilized on glutathione-agarose beads. Heparin and heparan sulfate (HS), but not dermatan sulfate, chondroitin sulfates A and C, hyaluronic acid, and K5 polysaccharide, competed with 3H-labeled heparin for binding to immobilized GST-Tat and inhibited HIV-LTR transactivation induced by extracellular GST-Tat. Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was >/=1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity.
1型人类免疫缺陷病毒（HIV-1）Tat蛋白从受感染细胞中释放出来。细胞外的Tat进入细胞后会刺激HIV长末端重复序列（LTR）以及内源性基因的转录活性。肝素可调节细胞外Tat的血管生成活性（阿尔比尼，A.，贝内利，R.，普雷斯塔，M.，鲁斯纳蒂，M.，齐切，M.，鲁巴泰利，A.，帕利亚隆加，G.，布索利诺，F.，和努南，D.（1996年）《癌基因》12卷，289 - 297页）和转录活性（曼，D.A.，和弗兰克尔，A.D.（1991年）《欧洲分子生物学组织杂志》10卷，1733 - 1739页）。在此我们证明，肝素能特异性结合以谷胱甘肽S -转移酶（GST）融合蛋白形式产生并固定在谷胱甘肽 - 琼脂糖珠上的重组HIV-1 Tat。肝素和硫酸乙酰肝素（HS），但不是硫酸皮肤素、硫酸软骨素A和C、透明质酸以及K5多糖，能与3H标记的肝素竞争结合固定化的GST-Tat，并抑制细胞外GST-Tat诱导的HIV-LTR反式激活。选择性的2 - O -、6 - O -、全O -去硫酸化或N -去硫酸化/N -乙酰化显著降低了肝素结合GST-Tat的能力。全O -去硫酸化和2 - O -去硫酸化的肝素在抑制GST-Tat反式激活活性方面的能力也有所降低。与传统的13.6 kDa肝素相比，极低分子量肝素结合GST-Tat以及抑制其LTR反式激活活性的能力显著下降。然而，当3.0 kDa肝素在固定化GST-Tat上进行亲和层析以分离结合和未结合亚组分时，与未结合组分相比，结合Tat的组分在抑制GST-Tat反式激活活性方面的效力高≥1000倍。结果表明，Tat与肝素/HS以大小依赖的方式相互作用，并且高亲和力的Tat-肝素相互作用至少需要一些2 - O -、6 - O -和N位被硫酸化。所测试的糖胺聚糖的Tat结合活性与其影响细胞外Tat反式激活活性的能力相关，这表明有可能设计出具有Tat拮抗剂活性的特定肝素/HS样结构。
