Small efficient cell-penetrating peptides derived from scorpion toxin maurocalcine

Abstract

Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.

FIGURE 1.

Efficacy of cargo penetration as function of grafting position on MCa_UF1–33. A, amino acid sequence of MCa_F in single-letter code. The positions of half-cystine residues are highlighted in red. Cys residues are numbered, and basic amino acids are highlighted in blue. Secondary structures (β-strands) are indicated by arrows. The gray box is the sequence of homology of MCa with the dihydropyridine-sensitive Ca_v1.1 channel. B, amino acid sequences of unfolded MCa analogues in single-letter code. Cys residues are replaced by isosteric 2-aminobutyric acid residues (Abu, in red) to form MCa_UF1–33. An additional N-terminal (C-MCa_UF1–33) or C-terminal (MCa_UF1–33-C) Cys residue was added in two novel analogues competent for cargo grafting (shown in gray). C, confocal microscopy images illustrating cell penetration of Cy5-C-MCa_UF1–33 and MCa_UF1–33-C-Cy5 (green labeling). Plasma membranes are labeled with concanavalin A-rhodamine (in red). CHO cells were incubated 2 h with 1 μm peptide concentration. D, comparison of cell penetration efficacy between Cy5-C-MCa_UF1–33 and MCa_UF1–33-C-Cy5 as determined by FACS. CHO cells were incubated for 2 h with 3 μm peptide, washed and treated 5 min by 1 mg/ml trypsin before quantification of intracellular fluorescence. a.u., arbitrary unit.

FIGURE 2.

Primary structure of truncated MCa_UF analogues and comparison of cell penetration efficacies. A, primary structures of truncated MCa_UF-C analogues and determination of their net positive charge and percentage of basic amino acid residues within the sequence. A total of 12 truncated MCa_UF-C analogues were produced (three with truncations in C terminus, seven in N terminus, and two in both N and C termini). Positively charged residues are in blue (His residues were not counted), whereas Abu residues that replace Cys residues are in red. B, comparative cell penetration efficacy of all MCa_UF-C-Cy5 truncated analogues that possess a net positive charge ≥ +5. Code colors: red (net charge +8), blue (+7), pink (+6), and green (+5). The nontruncated MCa_UF1–33-C-Cy5 analogue is shown as reference (black line) for the efficacy of cell penetration of all analogues. Experimental conditions: CHO cell incubation with 1 μm concentration of each analogue for 2 h and fluorescence quantification by FACS. a.u., arbitrary unit. C, same as B but for truncated MCa_UF-C-Cy5 analogues with positive net charge ≤ +2.

FIGURE 3.

All truncated MCa_UF-C-Cy5 vector-cargo complexes have resembling intracellular distributions. A, intracellular distribution of N-terminal truncated MCa_UF analogues. B, intracellular distribution of C-terminal truncated MCa_UF analogues. C, intracellular distribution of N- and C-terminal truncated MCa_UF analogues. CHO cells were incubated for 2 h with 3 μm MCa_UF-C-Cy5 vector-cargo complexes, before extensive washing, membrane labeling with rhodamine-conjugated concanavalin A, and live cell confocal microscopy imaging. Cy5 is in blue, and rhodamine is in red. White arrows illustrate a tendency for a preferential apical localization of the Cy5 dye. Yellow arrows illustrate the tendency for a subplasma membrane labeling of the Cy5 dye.

FIGURE 4.

Membrane staining is diffuse whereas intracellular staining is punctuated. A, lower magnification image of CHO cells stained with 3 μm MCa_UF14–25-C-Cy5 that illustrates a predominant subplasma membrane rim-like distribution. B, diffuse membrane staining of CHO cells by MCa_UF22–33-C-Cy5. White arrows indicate domains of the plasma membrane where the diffuse staining of the peptide-cargo complex is the most evident. C, extent of colocalization of the Cy5-labeled peptides with the rhodamine-labeled plasma membrane. NS, nonsignificant; *, ≤0.1; **, ≤0.05; and ***, ≤0.001.

FIGURE 5.

Amiloride sensitivity of truncated MCa_UF peptide cell entry. A, representative FACS analyses of the effect of 5 mm amiloride on MCa_UF1–33-C-Cy5 (upper left), MCa_UF1–15-C-Cy5 (upper right), MCa_UF20–33-C-Cy5 (lower left), and MCa_UF18–33-C-Cy5 (lower right) entries. Numbers in red represent average decrease or increase in peptide entry upon amiloride treatment. Cells were treated for 2 h with 3 μm peptide concentration with or without 5 mm amiloride. a.u., arbitrary unit. B, average effect of amiloride on mean cell entry of the truncated peptides. Positive values reflect increase in cell entries, whereas negative values indicate reduction in cell penetration.

FIGURE 6.

Dose-dependent cell penetration of truncated MCaUF peptides. A, representative example of the dose-dependent cell penetration of MCa_UF8–33-C-Cy5 in CHO cells as analyzed by FACS. The peptide was incubated for 2 h with the cells before analyses. There was no saturation of cell entry for a concentration up to 33 μm. B, dose-dependent cell penetration of N-terminal truncated MCa_UF peptides compared with MCa_UF1–33-C-Cy5 (open circles, dotted line). a.u., arbitrary unit. C, dose-dependent cell penetration of C-terminal truncated MCa_UF peptides. D, dose-dependent cell penetration of N- and C-terminal truncated MCa_UF peptides. Note the increase in scale for the penetration of these two peptides.

FIGURE 7.

Lack of pharmacological effects of the truncated peptides and reduced cell toxicity. A, effect of MCa_F, MCa_UF1–33, and truncated MCa_UF peptides on [³H]ryanodine binding. Data are expressed as -fold increase in binding induced by the peptides. B, effect of 1 and 10 μm MCa_UF1–33 and truncated MCa_UF peptides on CHO cell viability. Peptides were incubated for 24 h with the cells in vitro.