| As a common bacterial infectious disease, dental caries affects people’s oral health.The treatment methods commonly used to cure caries have several certain defects, which make people wish to seek new efficient natural products that have non-toxic side effects.It has been reported that plant polyphenol compounds is a natural functional ingredient to protect oral health. Procyanidins are polyphenol polymers that composed of catechins or catechin, its intervention effect on dental caries is 2 to 10 times higher than tea polyphenols, but the mechanism of how procyanidins effecting on caries is not very clear.Streptococcus mutants(S. mutants) has been implicated as one of the primary pathogenic bacteria that cause caries. The key to the occurrence of caries is the adhension and accumulation of S. mutants on teeth surface. And the process which protein from the surface of S. mutants interacting with the saliva acquired membrane receptor is supposed to be fundamental to the adherence of bacteria on teeth surface. Glucosyltransferases(GTFs) and glucan binding protein(GBPs) synthesized by S.mutants are crucial virulence factors to induce caries. S.mutan(Ingbritt c) can produce three kinds of GTFs, they are GTFB, GTFD and GTFC, particllarly GTFB can produce water-insoluble glucan which plays a pivotal role in promoting the adhesion of bacteria to teeth surface. The total length of gtf B gene is approximately 4.8 kb, The catalytic region(CAT) is located in the N-terminal of gtf B gene which encoding GTFB/CAT protein. GBPB as one kind of GBPs(GBPA, GBPB, GBPC and GBPD) is regarded as the "bridge" to the adhesion and accumulation of S.mutans on teeth surface, it not only has the function of combining with dextran, but is also relates to the cell wall formation and the activity of peptidoglycan from S.mutans. Our study use sorghum procyanidins(SPC) tetramers which isolated from sorghum testa to intervent carirs, we obteained GTFB/CAT protein and GBPB-sumo protein with high purity and high activity through gene cloning technology, and then adopt three spectroscopy technologies: fluorescence quenching, ultraviolet-visible(UV-vis) and circular dichroism(CD) spectra to research the interaction between two proteins and SPC tetramers.The main contents and conclusions are as below:1. Isolation and identification of SPC tetramersSPC-Ⅵ fraction isolated from sorghum testa was identified by RP-HPLC-MS/MS, it showed that SPC-Ⅵfraction is mainly consist of procyanidins tetramers.2. Gene cloning and expression of the catalytic region of gluscosyltransferasesa BThe PCR primes were designed according to sequences of gtf B gene from S.mutans UA159(Ingbritt c) published in the NCBI and the sequences at the ends of CAT in gtf B.The CAT of gtf B was amplified by PCR from the genome of S.mutans UA159(Ingbritt c), and then linked with the vector p ET-28b(+). The recombinant p ET-28b(+)-gtf B/CAT was transformed into Escherichia coli(E.coil) BL21, the best expressed conditions were 37 ℃ or 30 ℃, 4h, and the concentration of inducer(IPTG)was 1 mmol/L. The protein expressed in E.coil BL21 was found to be insoluble inclusion body, it was purified by Ni2+-NAT resin. The insolule inclusion body was renaturated by carbamide to restore solubleness, an expected 44 KDa water-soluble protein with 80%purity was identified by SDS-PAGE analysis. The activity and specific activity of GTFB/CAT were 131.67 m IU and 1.66 IU/mg respectively. The GTFB/CAT protein obtained by gene cloning was detected according to Nano LC-MS/MS, three trusted proteins was found from Streptococcus mutans serotype c(Strain ATCC 700610/ua159),the GTFB protein had got the highest score.3. Gene cloning and expression of glucan-bingding protein BThe gbpB gene was synthesized in vitro based on the sequences of gbpB gene from S.mutans UA159(Ingbritt c) published in the NCBI and has been codon optimized according to the E. coli preference. The gbp B was amplified by PCR from the genome of S.mutans UA159(Ingbritt c), and then linked with the vector p ET-28a-sumo. The recombinant p ET-28a-sumo-gbp B was diverted into into Rosetta(DE3), the expressed conditions were 37 ℃, 3h, 158 r/min and the concentration of IPTG was 0.5 mol/m L, the protein was found in expression supernatant and it was purified with Ni2+-NAT resin. Anexpected 66.2 KDa water-soluble protein with 90% purity was identified by SDS-PAGE analysis.The GBPB-sumo protein obtained by gene cloning was detected according to Nano LC-MS/MS, three trusted proteins was found from Streptococcus mutans serotype c(Strain ATCC 700610 /ua159), the GBPB protein had got the highest score.4. Spectroscopic studies on the interactions between SPC tetramers and GTFB/CAT, GBPB-sumo proteinsThe method Somogyi was used to measured the activity of GTFB/CAT protein that interacting without or with SPC tetramers, when the concentration of SPC tetramers was1.08×10-4 mol/L, the activity of GTFB/CAT protein decreased 94.24%from 131.67 m IU to 7.59 m IU. In our study, fluorescence quenching, UV-vis absorption and CD spectra were employed to characterize the interactions between SPC tetramers and GTF-I/CAT protein, GBPB-sumo protein. According to the Stern-Volmer equation, the quenching constant Ksv of SPC tetramers on GTF-I/CAT and GBPB-sumo were indentified as 2408.1M-1and 2346.6 M-1,the bimolecular quenching constant kq of two protein were 2.4081×1011 M-1·s-1 and 2.3466×1011 M-1·s-1. The binding constant KA were calculated as1522.6 M-1 and 2618.2 M-1 respectively, meanwhile the number of binding sites n were both 1. The results of fluorescence quenching experiments demonstrated that SPC tetramers have a high affinity for two proteins and the interaction is a single static quenching process. SPC tetramers also showed an effect on the UV-vis absorption spectra of two proteins with different red shift due to the changes in the micro-environment of the aromatic amino acid residues, which further demonstrated that a static quenching occurred between SPC tetramers and GTF-I/CAT protein, GBPB-sumo protein. The secondary structure of two proteins were both dramatically changed when SPC tetramers were combined with them. With the concentration of SPC tetramers increasing, the proportions of α-helix and random coil gradually decreased to 0.00 %, while that ofβ-sheet increased to 100.00%finally in both proteins, and β-ture only exist GBPB-sumo was also decreased to 0.00%. The secondary structural analysis by CD spectra suggested that SPC tetramers had broken hydrogen bond which maintain structural stability ofprotein, and the amino acid residues in the hydrophobic area of protein might be exposed by combining with SPC tetramers. In conclusion, SPC tetramers have effects on the structures or enzyme activities of two virulence proteins from S.mutans, which may further prevent the adhension and accumulation of bacteria on teeth surface and will finally intervene caries. |
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