Identification Of Novel Glycoproteins From Mycobacterium Tuberculosis H37Rv Culture Filtrate Protein By Affinity Chromatography And HPLC-MS

Currently, Mycobacterium tuberculosis is still a chiefly serious infectious diseases. According to the statistic from WHO, there are almost 800millions people are infected by tuberculosis and 300millions people die from tuberculosis in the world annually. Therefore, it is a important items for human being to learn how to prevent and how to control tuberculosis. Some investigations on other bacteria have shown that the bacterial glycoproteins are closely related to pathogenesis. Their functions involved the stability maintaining for the cell structure, recognize for call surface and cell inner; adaptation and sustaining for the cell biological and chemical character, such as the protein solubility ,surface charges. The identified mycobacterium tuberculosis glycoproteins are limited so far, to screen the novel glycoproteins and probe their function and synthesis will be great meaningful for totally understanding the mechanism of tuberculosis and developing new drugs or vaccine against tuberculosis.By screening the glycoprotein from different fractions in mycobacterium tuberculosis, such as cell wall, cell membrane, cytosol, and culture filtrate protein(CFP), people discovered that the glycoprotein only exist in CFP. Further more, studies on the synthesis of glycoprotein in mycobacterium tuberculosis indicated that only the substrate which can be translocated to the outside of the cell can be glycosylized by mannose transferase.On the contrary, the protein in the cytosol or membrane can be glycosylized .Based on these existing theory , we only choose the CFP, other than other fractions, as the target to screen the novel glycoprotein from mycobacterium tuberculosis.All the operation related mycobacterium tuberculosis H37Rv strains were proceeded in the biosafety grade 3 hood. The 50% glycerol stocked strains were inoculated on the 7H11 agar plates, then grew in 37℃incubator for 2 weeks. The single colony was then picked up and transfered to 10ml 7H9 broth, growing on shaker at 37℃for another 2 weeks, and then transferred the 10ml culture to 1L GAS media. The bacteria grew on the shaker with 100rpm at37℃. Almost ten days later, while the OD600 was in the window between0.6 - 0.8 ,then stopped growing, took out the culture.Harvest the culture by centrifuge with 3000rpm at 4℃for 15mins,resolve the pellet cell by 100ml GAS media (no tween)and then inoculated them to 10 separate 1L GAS media, kept on growing in 37℃for 2 weeks. 2 weeks later, stopped growing , took out the culture and settled down the cell ,move the most of the settled cell by 50ml pipet ,then filtrated the left culture by 0.2μm Zapcap filter, the filtrated fraction was collect by 3 4L sterilized bottles and got the crude CFP. The CFP was then moved to 20L Amicon reservoir in cold room. Concentrated the CFP by 3kDa filter membrane with the pressure from compressed Nitrogen .Kept the CFP Flowing into the filter chamber slowly and meanwhile kept the stir bar working to prevent the protein from precipitation. During this process, the protein will be blocked by the membrane if their size is bigger than 3kDa,finally, the crude CFP became concentrated CFP. Its volume will become almost 10～50ml.The concentrated CFP was then dialyzed against 10mM ammonium bicarbonate. Change the buffer every 8hours,totally 3 times. The protein concentration was detected by Biorad 680 microplate reader(wavelength 550Nm).diluted CFP protein by ConA binding buffer to 1mg/ml,then loaded sample on the column packed with ConA sepharose 4B resine. After washing, eluted the glycoprotein by elution buffer. During the purification, the flow rate was controlled by pump as 1ml/min.collected the elution fraction, and dialyzed it against 10mM annonium carbonate to remove sugar and salt in the sample. Detected the protein concentration by BCA method. The purified protein can be separated from each other by running 15% SDS-PAGE. The proteins molecular weight can be confirmed by coomassie staining. The different protein bands were cut from SDS-PAGE gel, and chopped to pieces. After destaining, extraction ,thesamples were digested by trypsin overnight in 37℃incubator. Upper fraction was taken after centrifuge, then dried sample by servant. Centrifuged again and discarded the pellet, then it was ready for running HPLC-MS.RP-HPLC was used to separated the peptides or glycopeptides according to their polarity. The sample used every time was only 5μl. The flowing phase included buffer A (TFA:H2O = 0.1:99.9) and buffer B(TFA:H2O:Acetonitrile = 0.1:9.9:90).the flowing rate was 5ul/min.The step elution procedure included that the 98% buffer A and 2% buffer B were used first,then slowly change to 8% buffer A and 92% buffer B.It took 1hour .The pH valueof the buffers was 7.4.The MS equipment used was ESI-Ion trap MS.the power was 4Kv and the CID was 35%.Software Xcalibur was used to evaluated the result of HPLC and the loss of hexose from peptide fractions.Currently, all the confirmed TB glycoproteins are glycosylated by mannose, the molecular weight of mannose is 162.So if a peptide molecular lose one or more mannoses, then its molecular weight will lose 162,324,486,648, et al; meanwhile, the ions can have 1 or more charges, so if mannoses were broken from peptide backbone, then its m/z will lose 162,81,54,40.5,et al. Accordingly, we can deduce the glycosylation from MS data by its change of neutral loss. Through rough analysis, among the six proteins from SDS-PAGE, only the 45kDa and 26kDa proteins were identified glycoproteins, the other four could not be found any supported informations as glycoproteins. On further study, the 45kDa was confirmed as same as the identified glycoprotein modD, so only 26kDa was chosen for analysis from MS data.In the mass spectrum, at the retention time of 24.65mins, one of the peptide from 26kDa was found regular neutral loss of 54 for m/z by using software Xcalibur. Its parent ion was 1467,its daughter ions were 1413.08,1459.19,1305.20 respectively, this appears the feature of manno-glycosylation. The amino acid sequence of this peptide were searched by software Bioworks against TB protein database and found as followed: HVPGHTGTPASGDLASL. From this sequence, there are two"T"and two"S", especially the second T the two S are all flanked by P or A, that is the mainly features for O-link glycosylation. Blast this sequence withexisting TB protein data base, there are totally four proteins were found, but whatever from the peptide probability, molecular weight, only the protein Cu-Zn superoxide dismutase(SodC) was matched. That means the SodC is a novel glycoprotein from mycobacterium tuberculosis.So far, the quantities of TB glycoproetin is quite a few, which limits the understanding of the synthesis pathway and function of glycoproteins, but it could not blocked the scientists'step to go forward for searching novel glycoproteins just because they probably have more important function than that in eukaryotic system. Certainly, it is a little difficult to find glycoproteins because they are always stained lightly on SDS-PAGE by coomassie, and the glycan can sometimes also effect the separation of peptide in RP-HPLC column. But anyway, in this study, by using ConA affinity chromatography combined with HPLC-MS/MS, a new 26kDa TB glycoprotein SodC was identified from TB CFP, this will be helpful for collecting glycoproteins information and enlarging the glycoprotein database for TB. It will be important for further study of TB glycoproteomics. With the more and more accumulated knowledge on synthesis, structure and function of TB glycoprotein, it would not be too far for human being to totally control and prevent Mycobacterium tuberculosis.