Ginsentide-Like Coffeetides Isolated from Coffee Waste Are Cell-Penetrating and Metal-Binding Microproteins

• DOI: 10.20944/preprints202308.1524.v1
• Publication Date: 2023-08-23T00:31:31Z
• Authors: James P. Tam; Jiayi Huang; Shining Loo; Yimeng Li; Antony Kam
• Keywords: Metal-Binding Peptides; Organic chemistry; Coffea; Non-Chitin Binding Hevein; Coffee; Article; metal-binding peptides; QD241-441; Microproteins; microproteins; Ginsentide; Cysteine-Rich Peptides; Cysteine; Disulfides; Micropeptides; cell penetration; :Biological sciences [Science]; Chemical Synthesis; 540; Cell Penetration; Hevein-Like Peptide; Science::Biological sciences; coffee waste product; Coffee Waste Product; chemical synthesis; cysteine-rich peptides

Coffee processing generates a huge amount of waste which contains many natural products. Here, we report the discovery of a panel novel cell-penetrating and metal-ion binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and database search, we showed that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif but without a chitin-binding domain. NMR determination of cC1a revealed a compact structure that confers it resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis revealed that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement like ginsentide precursors. Molecular modeling together with experimental validation revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, and which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have potential to target intracellular proteins.