Abstract
Homologous endopeptidases AlpA and AlpB are components of the secreted complex of lytic enzymes of the Gram-negative bacterium Lysobacter sp. ХL1. These enzymes are synthesized as precursors that consist of a signal peptide, propeptide, and proteolytically active mature part. To understand the topogenetic features of these proteins, bacterial cell fractions were investigated by a sensitive sandwich enzymelinked immunosorbent assay and immunoblot analysis with the use of monoclonal antibodies recognizing unique epitopes of proteins’ mature forms and their propeptides. Only mature forms of the enzymes, without propeptides, were shown to be released outside the cell into the environment. AlpA significantly exceeds AlpB in the production level at the early stationary growth stage. The AlpB precursor was revealed in the cytoplasmic and periplasmic fractions, and the AlpA precursor was found only in the cytoplasmic fraction. The periplasmic fraction was also found to contain the mature forms of both enzymes and their propeptides. These results indicate that AlpA and AlpB are released into the environment through different mechanisms. AlpA is translocated across the cell envelope without being interrupted in the periplasm. The homologous AlpB enzyme, on the contrary, accumulates in the periplasmic space and is captured by outer membrane vesicles in the process of their formation.
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Abbreviations
- ELISA:
-
enzyme-linked immunosorbent assay
- CF:
-
culture fluid
- LU:
-
lytic unit
- MA:
-
monoclonal antibodies
- MF:
-
PPF, and CF, membrane, periplasmic, and cytoplasmic fraction
- AEBSF:
-
4-(2-aminoethyl)phenylculfonyl fluoride
- ECL:
-
enhanced chemiluminescence
- PBS:
-
phosphate buffered saline
- PBST:
-
PBS containing 0.1% Tween 20
- ProA and ProB:
-
propeptides of AlpA and AlpB endopeptidases.
References
Kulaev, I.S., Stepnaja, O.A., Tsfasman, I.M., Tchermenskaja, T.S., Ledova, L.A., Zubrizkaja, L.G., and Akimenko, V.K., RF Patent no. 2193063, 2002.
Lapteva, Y.S., Zolova, O.E., Shlyapnikov, M.G., Tsfasman, I.M., Muranova, T.A., Stepnaya, O.A., Kulaev, I.S., and Granovsky, I.E., Appl. Environ. Microbiol., 2012, vol. 8, pp. 7082–7089.
Begunova, E.A., Stepnaya, O.A., Lysanskaya, V.Y., and Kulaev, I.S., Biochemistry (Moscow), 2003, vol. 68, pp. 735–739.
Vasilyeva, N.V., Shishkova, N.A., Marinin, L.I., Ledova, L.A., Tsfasman, I.M., Muranova, T.A., Stepnaya, O.A., and Kulaev, I.S., J. Mol. Microbiol. Biotechnol., 2014, vol. 24, pp. 59–66.
Vasilyeva, N.V., Tsfasman, I.M., Suzina, N.E., Stepnaya, O.A., and Kulaev, I.S., FEBS J., 2008, vol. 15, pp. 3827–3835.
Silen, J.L., Frank, D., Fujishige, A., Bone, R., and Agard, D.A., J. Bacteriol., 1989, vol. 171, pp. 1320–1325.
Fujishige, A., Smith, K.R., Silen, J.L., and Agard, D.A., J. Cell Biol., 1992, vol. 118, pp. 33–42.
Rudenko, N.V., Tsfasman, I.M., Latypov, O.R., Ledova, L.A., Krasovskaya, L.A., Karatovskaya, A.P., Brovko, F.A., Vasilyeva, N.V., and Stepnaya, O.A., Russ. J. Bioorg. Chem., 2014, vol. 40, pp. 272–278.
Karatovskaya, A.P., Rudenko, N.V., Tsfasman, I.M., Guseva, K.A., Laman, A.G., Boziev, K.M., Brovko, F.A., and Vasilyeva, N.V., Proc. Biochem. Soc., 2016, vol. 51, pp. 1521–1526.
Kudryakova, I.V., Suzina, N.E., and Vasilyeva, N.V., FEMS Microbiol. Letts., 2015, vol. 362, no. 18, pii: fnv137. doi 10.1093/femsle/fnv137
Cunningham, E.L. and Agard, D.A., Science, 2004, vol. 13, pp. 325–331.
Nossal, N.G. and Heppel, L.A., J. Biol. Chem., 1966, vol. 241, pp. 3055–3062.
Sitkin, B.V., Tsfasman, I.M., Stepnaya, O.A., and Kulaev, I.S., Biochemistry (Moscow), 2003, vol. 68, pp. 458–463.
Laemmly, U.K., Nature, 1970, vol. 227, pp. 680–685.
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Original Russian Text © N.V. Rudenko, A.P. Karatovskaya, I.M. Tsfasman, F.A. Brovko, N.V. Vasilyeva, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 5, pp. 501–506.
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Rudenko, N.V., Karatovskaya, A.P., Tsfasman, I.M. et al. Molecular forms of AlpA and AlpB lytic endopeptidases from Lysobacter sp. Xl1: immunochemical determination of their intra- and extracellular localization. Russ J Bioorg Chem 43, 526–530 (2017). https://doi.org/10.1134/S1068162017050119
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DOI: https://doi.org/10.1134/S1068162017050119