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bioRxiv · 10.1101/2024.03.04.583322

Exploring the molecular structure of lipids in the design of artificial lipidated antifungal proteins

Saputra, H.·Safaat, M.·Santoso, P.·Wakabayashi, R.·Goto, M.·Taira, T.·Kamiya, N.
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Abstract

Fungal infections have been a concern for decades, yet effective and approved antifungal agents are limited. We recently developed a potential method to enhance the antifungal activity of a small chitin-binding domain (LysM) from Pteris ryukyuensis chitinase A (PrChiA) by the site-specific introduction of a palmitoyl (C16) group catalyzed by microbial transglutaminase (MTG). Herein, we attempted the conjugation of a series of lipid-peptide substrates with LysM genetically fused with a C-terminal MTG-reactive Q-tag (LysM-Q) to yield LysM-lipid conjugates (LysM-lipids) with different lengths (LysM-C12, -C14, and -C16) and different numbers of alkyl chains [LysM-(C12)2, - (C14)2, and -(C16)2]. The enzymatic conjugation proceeded smoothly for all LysM-lipids, except for LysM-(C16)2 because of the low aqueous dispersibility of the hydrophobic (C16)2 lipid-peptide substrate. The combination of amphotericin B (AmB) with LysM-C14 or LysM-C16 exhibited the highest antifungal performance against Trichoderma viride whereas alterations in the number of alkyl chains were not effective in enhancing the antifungal activity of the LysM-lipids. Fluorescent microscopic analysis showed that the fungal cell wall was stained with C14- and C16-modified LysM-muGFP fusion proteins when combined with AmB, suggesting a synergistic action of AmB and LysM-lipids with a suitable lipid length. All LysM-lipids showed minimum cytotoxicity toward mammalian cells, suggesting that LysM-lipids could be a safe additive in the development of new antifungal formulations.

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Saputra, H., Safaat, M., Santoso, P., Wakabayashi, R., Goto, M., Taira, T., Kamiya, N.. 2024-03-08. Exploring the molecular structure of lipids in the design of artificial lipidated antifungal proteins. https://doi.org/10.1101/2024.03.04.583322

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