Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide’s ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.


GMA4C-GFP
Primer 4 R: 5'-GCGACGAAAACCACGGCAACGGCCAGAACCATGGTGATGGTGAT GGTGAGAAG-3' Recombinant GFP with GMA4C at either the N-or C-terminus (GMA4C-GFP or GFP-GMA4C, respectively) was cloned into the pET28 vector and expressed in E. coli BL21 (DE3) cells.All plasmids feature an N-terminus Hisx6 tag. Figure S1A illustrates the expressed constructs. Figure S1B shows the structural model of the proteins.Detailed protein sequences are found in Table S2.

Supplementary Figure S2
To assess GMA4C's capacity for cell penetration across different cell types, we subjected HGF cells to various concentrations of FITC-GMA4C.After two hours of incubation and thorough washing, we examined the cells using confocal microscopy imaging.Figure S2

Supplementary Figure S3
To investigate the intracellular protein uptake facilitated by the GMA4C peptide across various cell lines, we treated C2C12 myoblasts with either 5 μM of FITC-GMA4C peptide or GFP-tagged proteins for 2 hours.Subsequently, the cells underwent three PBS washes and fixation using 4% PFA.Finally, they were mounted on a glass slide for evaluation through confocal microscopy.Consistent with the results presented in Figure 3, the fluorescence microscopy images herein further confirm the efficient cellular uptake of FITC-GMA4C.Additionally, these images reveal partial colocalization with the endosomal marker Cav-1.A similar outcome was observed in HeLa cells (Figs. 5 and 6), where GMA4C effectively demonstrated its competence in internalizing GFP cargo into the cells.Measurement of protein penetration levels.The table summarizes Pearson's coefficient and the cellular uptake percentage of GMA4C-GFP.The latter was calculated based on the number of green pixels that colocalized with the cellular margins of red pixels (tubulin) and were above the threshold in both channels.The Pearson's coefficient for channel-1 (green) vs. channel-2 (red) was measured using the "Color 2" plugin in Fiji and is presented as the mean ± SD of duplicates.

Figure S1 .
Figure S1.Expression of recombinant GFP protein with N-and C-terminus GMA4C tag.(A) Illustration of protein constructs.(B) Comparison of AlphaFold models of GFP-tagged proteins with the GMA4C peptide.Surface structure representations created with ChimeraX software.
presents a series of images, including representative brightfield, DAPI (blue), FITC (green), and merged images.The images show the accumulation of FITC-GMA4C within the cells.

Figure S2 .
Figure S2.FITC-GMA4C Penetration into HGF Cells.(A) HGF cells were cultured for two hours with varying concentrations of FITC-GMA4C and subsequently stained with DAPI.Confocal microscopy images were captured to visualize FITC-GMA4C (green) and DAPI (blue) in cells attached to a slide.Scale bar: 100 µm.(B) Mean green fluorescence intensity relative to non-treated cells and normalized to the cell count.The signal was quantified using the Color Threshold tool in Fiji.Significance levels are denoted as follows: *p<0.05,**p<0.01,***p<0.005.

Figure S3 .
Figure S3.Facilitation of GFP Protein Delivery by GMA4C in C2C12 Cells.(A) Partial colocalization of FITC-GMA4C and GMA4C-GFP in C2C12 cells.Cells were incubated with 5 μM of FITC-GMA4C or GMA4C-GFP for two hours, fixed, mounted, and scanned by a confocal microscope.The images visualize FITC or GFP (green), DAPI (blue), and Cav-1 (red).Scale bar: 100 µm.Colocalization is indicated when similarly shaped structures appear yellow in the Merge.(B) Representative figure of GMA4C-GFP taken with a 63x Objective Lens.Scale bar: 100 µm.(C) Confocal microscopy images were captured to visualize GFP tagged with the GMA4C peptide (green) and DAPI (blue) in cells attached to a slide.Scale bar: 100 µm.(D) Mean green fluorescence intensity relative to non-treated cells and normalized to cell count.Measurement values were obtained using the Color Threshold tool in Fiji.

Figure S6 .
Figure S6.Cellular Delivery of GMA4C-GFP in HeLa Cells.(A) HeLa cells were incubated with variable concentrations of GMA4C-GFP for 2 hours.Cells were subsequently stained and imaged for DAPI (blue), GMA4C-GFP (green), and β-tubulin (red), and the cellular uptake of the protein was analyzed.Representative fields were imaged at 40× magnification.Scale bar: 100 µm.(B) Quantification of confocal image signal intensity.The bars represent the mean green fluorescence intensity relative to non-treated cells, normalized to the cellular surface area measured by β-Tubulin.Values were measured using the Color Threshold tool in Fiji.The values represent the mean ± SD of four replicates.(C)