| Tuberculosis is an ancient bacterial disease, which is caused by Mycobacterium tuberculosis infection or long-term latent infection, seriously damage to the human and animal health. About two billion people are latently infected with Mycobacterium tuberculosis around the world, it is responsible for approximately two million deaths each year and remains a leading cause of morbidity and mortality worldwide.The secreted proteins of Mycobacterium tuberculosis were separated and collected through liquid chromatography by Eui-Hong Byun et al in 2012, and the DC cells were processed with different tube proteins. They found that Rv0315 can significantly induce DC maturation via activating NF-κB and MAPK signal pathway, suggesting a novel potential immune antigen.In order to solve the present situation that extensively drug resistant strains of Mycobacterium tuberculosis continue to emerge and the efficacy of the current TB vaccine is limited, we need to explore the characteristic of the new Mycobacterium tuberculosis vaccine candidate. Thus, the structure and function research on Rv0315 is carried out. We resolved the three-dimensional structure of Rv0315 for the first time by the platform for structural biology, further more, we explored and discussed the function of Rv0315 and the mechanism of its function. The concrete research content is as follows:Rv0315 belongs to the β-1,3-glucanases family. The β-1,3-glucanases, which possess the highly sequence and structure similarity, belong to glycoside hydrolase 16 family(GH16). Based on the commonness, the amino acid sequence of β-1,3-glucanase whose structure and function is known were recovered from NCBI database first, and used them to create the sequence alignment with Rv0315. Rv0315 shows highly sequential similarity to GH16 family endo-β-1,3-glucanases, two conserved glutamates and the calaium binding sites also exist in Rv0315, the sequence alignment results suggest that Rv0315 is a β-1,3-glucanase.The high structure homology is another characteristic of GH16 family β-1,3-glucanases. Based on the structure characteristic of β-1,3-glucanases, we first construct the full-length expression plasmid p ET42b-Rv0315 of H37 Rv strain Rv0315, and then obtain the soluble protein using the E.coli expression system. We get a crystal with a high resolution of 1.7? through screening with the laboratory existing crystal screen kit and optimizing. The data were collected and the structure of Rv0315 was solved using the molecular replacement method. The structure of Rv0315 suggested that it conforms to the structure characteristic of β-1,3-glucanases. The overall structure comprises 16 β-strands forming two twisted β-sheets, the twisted β-sheets form a concave, which is the catalytic activity center; it also contains two short ɑ-helices and one calcium ion. Rv0315 is a β-1,3-glucanases base on its sequence and structure.Rv0315 is a inactive β-1,3-glucanase. The β-1,3-glucanases produced by bacteria, belong to GH16 family. All the β-1,3-glucanases could hydrolyze the glucans with 1,3-β-linkages, such as laminarin, some of them hydrolyzed β-1,3-1,4-glucosyl linkages, such as β-D-glucan from barley. As Rv0315 is a β-1,3-glucanase, then its hydrolytic activity against laminarin and β-D-glucan from barley was measured by DNS method. Strangely, Rv0315 is ineffective for the degradation of laminarin, and has no detectable activity toward β-D-glucan from barley. All the results above indicate that Rv0315 is an inactive β-1,3-glucanase. We analyzed the phylogenetic tree of GH16 family β-1,3-glucanases whose structure and function is known. The results show that Rv0315 and the β-1,3-glucanase from Mycobacterium marinum(Mm4PQ9) formed an independent clade. Although the structure of 4PQ9 was resolved, the function of it is still unknown. The related research has annotated it as a β-1,3-glucanase. So we synthesized the 4PQ9 gene and successfully obtained the soluble protein, then detected the hydrolysis activity of 4PQ9 with the same method. The results showed that 4PQ9 is also an inactive β-1,3-glucanase. Therefore, We found an independent clade of GH16 family β-1,3-glucanases, and the clade was composed of two inactive β-1,3-glucanases.Why Rv0315 has no detectable hydrolysis activity towards polysaccharide? The mechanisms of Rv0315 with no hydrolysis activity was explained by comparing the structure of Rv0315 with β-1,3-glucanase from Z. Galactanivorans(PDB number 4BOW). 4BOW showed high catalytic activity against laminarin, meanwhile it can hydrolyzed MLG which is a β-1,3-1,4-glucanase and like β-D-glucan from barley. Through the structure comparsion, we found that these residues related to binding polysaccharide at subsites-2 and-3 of 4BOW are conserved in GH16 family β-1,3-glucanase, but not existent in Rv0315. The residues related to binding glucose unit at subsites-1 are also conserved in Rv0315. So presently the only known molecule that can bind Rv0315 is ethylene glycol. Ethylene glycol molecules were found in Rv0315 structure, and was bound to subsite-1 of the catalytic cleft Rv0315, mimicking a bound glucose moiety. As can be seen from the whole structure comparison, Rv0315 has a large open groove. We can draw a conclusion from these results that Rv0315 lacked the residues related to combine with substrate resulting in a large cavity, which led to cannot firmly bind the polysaccharides and displayed no detectable hydrolytic activity towards the polysaccharides. We further performed the ITC experiments to confirm that laminarin cannot bind to Rv0315, while 4BOW-E269 S can bind laminarin and form a good fitting curve. One molecule of the laminarin can combine with two molecules of 4BOW-E269 S.The relationship bewtween the structure of Rv0315 and its immunological function. Rv0315 is an inactive β-1,3-glucanase, but the two glutamates playing a role in the catalytic residues is reserved in Rv0315. Rv0315 may lose the function of hydrolysis of polysaccharides but still participate in immune function through the long-term evolution. Because Rv0315 has a large open groove, it may be more likely to be exposed to the immune system to become the target of the immune system and thus play a role in determining the adaptive immune response to TB via the immune molecules binding to Rv0315. In order to verify our speculation, we detected the activation of the NF-κB signaling pathway by Rv0315, Rv0315E176 S and Rv0315D289 A using luciferase reporter assays. The results indicated that Rv0315 and Rv0315D289 A can activate the NF-κB signaling pathway in a dose dependent manner, whereas the substitution for catalytic site Glu-176 cannot activate the NF-κ B signaling pathway. In addition, we also use different concentrations of Rv0315 and Rv0315 mutants process the DC cells and detect the DC maturation, the results consistent with the results of luciferase reporter assays. Rv0315 and Rv0315E176 S can induce DC maturation, whereas Rv0315E176 S cannot.In conclusion, Rv0315 may diverge from the GH16 family β-1,3-glucanases, and the inactive member offers new insights into the GH16 family β-1,3-glucanases. Our study reveals that an inactive β-1,3-glucanase in Mycobacterium tuberculosis drives T-helper 1(Th1) cells immune responses, and Glu-176 is the key amino acid involved in immune function, which may provide a new basis for mycobacterium tuberculosis vaccine development. |
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