Study Of The Interaction Between Glycosaminoglycans And TAT Peptides

Acquired immunodeficiency syndrome(AIDS)is caused by human infection with human immunodeficiency virus(HIV).According to the World Health Organization,by the end of 2016,there were approximately 36.7 million HIV infected persons in the world including 1.8 million increase in 2016.Up to now,AIDS has already caused more than 35 million deaths.In 2016 alone,there were more than 1 million people died of HIV-related illnesses in the world.Therefore,overcoming AIDS is an urgent problem to solve.HIV virus is divided into type ? and type ?,the former is the current popular type.Transactivator of transcription(TAT)is an trans-activating protein encoded by the human immunodeficiency virus ?(HIV-?)genome and is a necessary factor for the replication and expression of HIV-? genes.TAT protein participates in the initiation of HIV transcription and the extension of the RNA chain by combining the transactivating responsive element(TAR)of the HIV Long Terminal Repeat(LTR).In addition to the effect as the activator of HIV-? transcription,TAT also exerts several important functions on infected and uninfected cells.It is released by infected HIV-?cells and taken up by neighboring uninfected cells.In the infected CD4+T lymphocytes,TAT protein could activate the replication and expression of the virus.But for uninfected T cells,TAT protein can be activated to make it easy to infect the virus and induce its immunosuppression and apoptosis.Therefore,the extracellular TAT protein play a key role in the pathogenesis of AIDS and in the process of infection HIV-? immune disorder.TAT protein and its mediated cell pathway may be a potential target to develop new anti-HIV vaccines and therapeutic approaches against AIDS.It was found that the cell surface of anionic proteoglycans,especially heparan sulfate proteoglycans,play a key role in the cellular uptake of TAT peptide.In HS-defective cells or application of heparin in the cell culture,the uptake of TAT protein was severely inhibited.Once the HS from the cell surface was removed by the heparanase,the TAT peptide was inhibited largely to enter the cells.Cells pretreated by glycosaminoglycans showed a huge fluorescence intensity drop inside the cells.All these studies suggested that the cellular uptake of TAT protein could be mediated by electrostatic interactions between the cell surface of negatively charged proteoglycans receptors and TAT protein.However,there are no well-known agreement on which the specific kinds of glycosaminoglycan molecules or structures were essential in the electrostatic interactions.Therefore,it is necessary to study the structure-activity relationship of the interaction between the glycosaminoglycans and the TAT protein.This project aims at studying the interactions between glycosaminoglycans and TAT peptides to find targeted inhibitors to prevent the entry of extracellular TAT proteins into cells and to provide a new treatment tool for AIDS and related disorders.Firstly,the interactions between various glycosaminoglycans and their oligosaccharides and TAT peptides were studied by the gel mobility shift assay(GMSA).It was found that heparin,heparan sulfate(HS),dermatan sulfate(DS),and chondroitin sulfate A/C(CS A/C)but not hyaluronic acid(HA)could interact with TAT peptides.We also found that HEP-dp4 and DS-dp8 were the minimal effective fragments to interact with TAT peptides for the first time.It was preliminarily confirmed that the amino acid sequence of TAT peptides and the sequence of sulfate groups of glycosaminoglycans played key roles in the interactions.The NS and 2S of GAGs played a decisive role in their interactions and N-acetylation could promote it,while the 6S had a little effect on it.Then,the cellular uptake assay indicated that all selected GAGs had a certain inhibitory effects on the TAT peptides from entering cells.The larger the molecular weight and the higher the degree of sulfation resulted in the more significant on the inhibitory effect.Finally,NMR was used to study the specific binding sites of GAGs involved in their interactions.IdoA2S was the essential binding site,2S and NS were also necessary for the interactions and the N-acetylation could promote their interaction,while the 6S had a little influence.