Coli Research The Prothymosin ¦Á N ~ ¦Á-terminal Acetylation Modification Mechanism

Post-translational modifications play an important role in organism, which make proteins have more complex construction, more complete functions, and more refined regulation. These modifications are protein glycosylation, N-terminal acetylation, phosphorylation, and C-terminal amidation and so on. N-terminal acetylation is one of the most widespread covalent modifications in eukaryotes, but rare in prokaryotes.N-terminal acetylation of proteins is catalyzed by N-terminal acetyltransferases (NATs) , which transfer CH3CO- (43 Da) from AcCoA to the alpha amino group of the N-terminal amino acid residues. A compatible N-terminal amino acid sequence and an appropriate NAT are involved in the process of proteins N-terminal acetylation. N-terminal acetyltransferase RimJ, RimL, and RimI are the known NATs in Escherichia coli, which take the responsibility of N-terminal acetylation of S5, L12 and S18 respectively.Thymosin alpha 1 (Tα1), an immunomodulator, origin from Prothymosin alpha (ProTα), is composed of 28 amino acid, and N-terminal acetylated. Tα1 is safe and effective treatment for viral hepatitis and tumors. The chemical synthetic Tα1"Zadaxin", is widespread in the treatment of HBV, HCV and non-small cell lung cancer. The method of biosynthetic Tα1 could reduce the cost and increase yield, but also provide a study model for the N-terminal acetylation of prokaryotes.Our previous work indicated that ProTαcan be partially N-terminal acetylated when expressed in E. coli DH5α. Fang's study indicated that RimJ took the responsibility of the N-terminal acetylation of Tα1-L12. In order to investigate the effect of the three Nα-terminal acetyltransferases (NATs) of E.coli on human prothymosinα(ProTα) acetylation, I constructed a series of defective E. coli strains (DH5αΔrimJ, DH5αΔrimL, DH5αΔrimI, DH5αΔrimJΔrimL, DH5αΔrimJΔrimI, DH5αΔrimLΔrimI, DH5αΔrimJΔrimLΔrimI)by knocking out three Nα-terminal acetyltransferase genes (rimJ, rimL and rimI), and expressed proTαin these defective strains. The acetylation level of ProTαproduced in defective strains was detected by RP-HPLC. And the results showed that the acetylation level differed from each other. ProTαwas completely unacetylated while rimJ was knocked out, which suggestes that rimJ gene is also associated with the acetylation of ProTαin the wild type DH5α.The relationship between Rims and the N-terminal acetylation level was investigated by mean of gene overexpression. ProTαwas completely acetylated when rimJ or rimL were over-expressed in DH5αand DH5αΔrimJΔrimLΔrimI. But, the acetylation of ProTαwas inhibited when rimI was over-expressed. Then the effect of RimI on ProTαacetylation was further researched. SAS program analysis confirm that: Over-expression of rimI, long term induction and the cross-effect could affect the level of N-terminal acetylation of ProTα(P<0.0001); The increasing N-terminal acetylated ProTαcould be observed after inducing 6h, whether RimI was over-expressed or not (P<0.0001). There were 37.4% N-terminal acetylated ProTαin the presence of overdose RimI, 64.3% N-terminal acetylated ProTαwithout of overdose RimI after inducing 12h, and we could observe the inhibition after inducing 6h (P=0.0007).N-terminal amino acid sequence can affect the acetylation level of protein. I carried out primary study about the effect of N-terminal amino acid sequence on the acetylation level of ProTα. I constructed a series of ProTαmutations with different N-terminal amino acid sequence. The MALDI-TOF MS result indicated that there was no change when the first amino acid Ser was replaced by Thr, and reduced N-terminal acetylated ProTαwas found when Ser was changed to Ala; ProTαwas completely unacetylated when Ser was changed to Gly; The initial methionine was not completely incised when Ser was changed to Met. There was N-terminal acetylated ProTαwhen the second amino acid Asp was changed to Glu and Val, and N-terminal degradation when the Asp was mutated by Val, Cys, Ala, and Pro, but the mechanism was unknown.Above all, rimJ gene is responsible for the N-terminal acetylation of ProTαin the wild type DH5α. ProTαcan be comletely acetylated when rimL or rimJ was over-expressed, but the acetylation of ProTαwas inhibited when rimI was over-expressed. Inaddition, N-terminal amino acid sequence could influence the level of N-terminal acetylation of ProTα. This study served as a model for the study of N-terminal acetylation in prokaryotes.