An 82-residue-long chimeric peptide was synthesised by solid phase peptide synthesis (SPPS), following the Fmoc protocol. Microwave (MW) radiation-assisted synthesis was compared to standard synthesis using low loading (0.20 mmol/g) of polyethylene glycol (PEG) resin. Similar synthetic difficulties were found when the chimeric peptide was obtained via these two reaction conditions, indicating that such difficulties were inherent to the sequence and could not be resolved using MW; by contrast, the number of coupling cycles and total reaction time became reduced whilst crude yield and percentage recovery after purification were higher for MW radiation-assisted synthesis.
This work was aimed at studying the Mycobacterium tuberculosis H37Rv Rv3494c protein, taking into account that it belongs to the mammalian cell entry family (mce) which is thought to have important functions in the disease's pathogenesis. The protein was characterized in silico; its presence on mycobacterial surface was confirmed by immunoelectron microscopy. High-activity binding peptides (HABPs) were identified by binding assays with (125)I; their ability to inhibit mycobacterial entry to two cell lines (U937 alveolar macrophages and A549 epithelial cells) was ascertained and their role in bacterial entry was confirmed by fluorescent microsphere internalization assay. This protein's predicted alpha-helix structure was confirmed by circular dichroism of its peptides. All HABPs inhibited mycobacterial entry to cells and that the 38379 peptide ((201)IDQAGPFLQAQIRAGGDIKSY(220)) had high binding ability and inhibited the mycobacterial entry to both cell lines assayed here. Rv3494c peptides 38370 ((21)LSVMAIFYLRLPATFGIGTY(40)), 38373 ((81)HMRLNSGTAIPSNVTATVRSY(100)) and 38379 ((201)IDQAGPFLQAQIRAGGDIKSY(220)) showed to be HABP and inhibited mycobacterial entry to A549 cells and peptide 38382 ((261)RPSFPALAASLANLGRVGVIY(280)) bind to U937 and inhibited the mycobacterial entry to this cell line; all of these sequences play an important role in cell line recognition and invasion, and may thus be considered in the search for prophylactic candidates against tuberculosis.
Like Thomas Hardy's famous novel Far from the Madding Crowd, Plasmodium falciparum parasites display their most relevant survival structures (proteins) involved in host cell invasion far away from the immune system's susceptible regions, displaying tremendous genetic variability, to attract the immune response and escape immune pressure. The 3D structure localisation of the conserved amino acid sequences of this deadly parasite's most relevant proteins involved in host cell invasion, as well as the location of the highly polymorphic, h i g h l y i m m u n o g e n i c r e g i o n s , c l e a r l y demonstrates that such structures are far apart, sometimes 90° to 180° opposite, thereby rendering the immune response useless. It is also shown here that these conserved, f u n c t i o n a l l y -r e l e v a n t s t r u c t u r e s a r e immunologically silent, since no immune response has been induced.
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