Once the presence and transcription of Rv0679c was determined Epacadostat mouse in the MTC, the next step consisted in evaluating protein expression by Western blot analysis of M. tuberculosis H37Rv sonicate. Goat anti-Rv0679c peptide serum detected two bands of about 18 and 20 kDa, which differ from the theoretical
molecular mass of 16.6 kDa predicted based on its amino acid composition. This slight difference could be caused by the post-translational modifications that lipoproteins undergo before reaching their destination as mature proteins, considering that pro-lipoproteins tend to be 2-3 kDa larger than mature lipoproteins . According to bioinformatics predictions, Rv0679c lacks of transmembrane regions and contains an N-terminal signal sequence as well as a SPAse II cleavage site between
residues 32-33, as indicated by the presence of a “”lipobox”" motif [LAGC] between amino acids 30-33. The presence of a signal peptide detected by using SignalP suggests that this protein is secreted via the Sec-dependent pathway, and is probably targeted by the lipobox motif to membrane surface where it remains attached by hydrophobic interactions. Briefly, after Rv0679c is translocated across the cytoplasmic membrane, the Cys residue of the lipobox motif is linked to a diacylglyceryl moiety. Then, a signal II peptidase cleaves off the signal peptide and the protein is anchored to the mycobacterial membrane via the diacylglyceryl moiety . These computational predictions are in agreement with the cellular localization observed in IEM studies in which the protein was detected on the surface of M. tuberculosis H37Rv bacilli. To determine Defactinib research buy whether the peptides comprising Rv0679c established ligand-receptor interactions with M. tuberculosis susceptible human host cells, binding assays were performed with the U937 phagocytic and A549 epithelial cell lines. HABPs 30985 to 30987 comprising amino acids 121-165 showed higher binding activities to receptors
on the surface of epithelial cells, whereas their binding activities to the phagocytic line were lower. Such differential binding behavior may be caused by differences between the surface receptors expressed by each http://www.selleck.co.jp/products/pembrolizumab.html cell line or their distinct physiological functions. Interestingly, Rv0679c HABPs 30985, 30986 and 30987 are consecutively positioned within the PP2 chemical structure protein’s C-terminus, suggesting that the region formed by these three HABPs is implicated in binding of M. tuberculosis to target cells. Also, the Hill analysis showed high binding affinity interactions with a large number of receptor molecules on the surface of U937 cells, as indicated by their dissociation constant within the nanomolar range. Moreover, the formation of ligand-receptor complexes appears to facilitate binding of more HABPs, as shown by the positive Hill coefficient. All HABPs tested in invasion inhibition assays prevented cell invasion by M. tuberculosis by a larger or comparable percentage, compared to the colchicine and Cytochalasin D controls.