engleski

Characterisation of peptidoglycan-based immunomodulators by NMR spectroscopy and molecular modelling

Peptidoglycan (PGN), is a unique component of the bacterial cell wall and is not only a well-known target for antibiotics, but is also capable of triggering the body’s immune defence when recognized by the Pattern Recognition Receptor (PRR) of the innate immune system, such as the Toll-like receptor 2 (TLR2) with CD14 as co-receptor, the nucleotide-binding oligomerization domain -1 and -2 (NOD1 and NOD2) and the family of peptidoglycan recognition proteins (PGRP). Involvement of the macrophage mannose receptor (MMR) in PGN recognition is indicated by immunological responses of mannosylated derivatives are the ligand binding profile of MMR: attachment of a hydrophilic moiety such as mannose to the monomeric unit of PGN (ManPGN) was found to decreasethe immunostimulating effects as well as switching the type of immune reaction. Thus these findings may point the involvement of the macrophage mannose receptor (MMR) in PGN recognition or alternatively a drastic change in the conformation of PGN derivative caused by the hydrophilic substituent might hinder/prohibit the binding of ManPGN to known receptors. As a first take on investigating this hypothesis, we have carried out a comparative study of the solution state conformations of PGN derivatives using NMR spectroscopy and molecular modelling. We have compared the monomeric unit of Dap-type PGN (PGM) is a non-toxic, non-pyrogenic immunostimulator and it mannosylated derivative (ManPGM). Ensembles obtained by NMR-restrained structure calculations on PGM confirmed that the glycan part exists in well defined conformations, while the peptide part is less ordered. The mannosylated derivatives sample a larger conformational space in solution than that of the parent PGM: the disaccharide part of the molecule show similar preferences in the orientation of the interglycosidic linkage as in PGM, while the pentapeptide part is loosely organized via transient H bonding interactions. The conformation of the mannosylated PGN compounds are therefore not significantly different that of the parent PGM molecule. These findings thus argue against the idea of conformational hindrance of receptor recognition. We have also studied the molecular recognition of a mannosylated peptidoglycan derivative by the model receptor Concavaline A (Con A). The derivative investigated contained a dipeptide fragment of conjugated with an adamantyl as well as to mannosyl group. Adjuvant effect of the investigated compound has been investigated and found to be comparable to that of PGM. The binding studied carried with NMR spectroscopy has shown that the mannosylated adamantylated tripeptide is weakly binding to Con A as evidenced by STD NMR signals, thus its recognition by MMR in the cell membrane of immune cells might be possible.

Peptidoglycan monomer; macrophage mannose receptor; mannose; immunomodulator; NMR spectroscopy

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