Enzymatic Preparation And Activity Study Of Heparosan Sulfated Derivatives

Heparin is widely used in the clinical therapy as an anticoagulant drug.Because the 2008 contamination crisis of animal origining heparin occurred in the United States,which led to some death cases,enzymatic synthesis of heparin from microbial heparosan became a hot topic.Heparosan is microbial capsular polysaccharide and could be produced by E.coli K5 strain via fermentation process.Thus,it is the suitable precursor of enzymatic synthesis of heparin and heparan sulfate(HS).It was already reported that heparin and heparan sulfate exhibited the acticity of anti-coagulant,anti-viral,and inhibit bacterial adhesion.However,less pubilicaiton was found about the anti-coagulant and anti-viral activity of heparosan sulfated derivatives via enzymatic synthesis from microbial heparosan.In this study,a recombinant strain was constructed to prepare 3-O-sulfotransferase-1(3OST-1),which was further utilized for 3-O-sulfated modification of NSNAH(N-sulfo,N-acetyl heparosan)to botain the final product NSNA3SHp(N-sulfo,N-acetyl,3-O-sulfoheparosan).Finally,the anti-HBV(hepatitis B virus)activity of NSNA3SHp was evalated.N-deacetylation and N-sulfonation of heparosan could be achieved via trimethylamine-sulfur trioxide chemical method.In this study,N-sulfo,N-acetyl heparosan was successfully prepared and its degree of N-sulfonation was calculated as 86.9%based on the relative integral values of different sugar residues in ~1H-NMR spectrum,suggesting it is suitable substrate for futher sulfated modification.Meanwhile,3-O-sulfotransferase-1(3OST-1)gene was successfully cloned from the wistar rat brain.After sequencing and BLAST alignment,it was found that the homology of 3OST-1 gene fragment and 3OST-1gene of rattus norvegicus reported by NCBI was 99%.Then the 3OST-1gene fragment was inserted into E.coli expression vector pET-28a to construct a recombinant plasmid pET-28a-3OST-1,which was successfully induced in E.coli(DE3).Soluble protein was obtained and its molecular weight was caculated as 39.49 kDa via UN-Scan-it gel 6.1.After purified by Ni-NTA affinity chromatography,the specific activity of the purified enzyme was 110.20 U/mg.Then sulfotransferase 3OST-1 was successfully expressed in large scale and utilized to sulfate NSNAH to prepare NSNA3SHp,the~1H-NMR spectrum analysis of which showed that the sulfate group was successfully transferred to the specific site.During anti-viral activity study,0.37μg/mL of NSNA3SHp showed no cytotoxicity on the human hepatoma cell line(hepG22.1.5).What’s more,it shows a little bit anti-viral activity on HBV.Others showed cytotoxicity on the hepG22.1.5,so the results were unreliable and could not determine whether it showed anti-viral activity on HBV.