Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins

Abstract

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Keywords: VyPAL2; biocatalysts; cell surface labeling; protein cyclization; protein labeling.

Scheme 1

A general scheme of VyPAL2-mediated intramolecular and intermolecular ligation reactions. NGL (Asn-Gly-Leu) = a C-terminal recognition motif for VyPAL2. POI 1 and POI 2 = protein of interest 1 and 2.

Scheme 2

Examples of VyPAL2-mediated ligation (VML) demonstrated in this study.

Figure 1

VyPAL2-mediated protein C-terminal labeling with a fluorescent peptide. See Supporting Information for detailed experimental conditions, procedures, and analytical data. All yields reported in this study are conversion yields based on HPLC analysis.

Figure 2

C-terminal labeling of Z_EGFR 3 by fluorescent peptide 5 for cell binding analysis. (A) The labelling reaction scheme. (B) Monitoring of the reaction by HPLC and SDS gel (upper panel; note: the band corresponding to peptide 5 on the Coomassie gel image was washed away by the destaining solution during the destaining step), and ESI-MS analysis of the labelling product (lower panel; 3: Calcd. 9014.1, Obsvd. 9016.2; 8: Calcd. 9883.5, Obsvd. 9885.9). (C) Fluorescent microscopy imaging and flow cytometry analysis. Fluorescence-labeled affibody 8 and ubiquitin 6 (Figure S2) were used to treat cancer cell lines MCF-7 (EGFR-low expressing) and A431 (EGFR-overexpressing), and the treated cells were subjected to flow cytometry analysis and fluorescent microscopy. As shown in (C), only 8 was able to bind on the EGFR+ A431 cells. As the negative control, 6 bound to neither MCF-7 nor A431 cells.

Figure 3

VyPAL2-mediated inter-protein ligation. (A) Schematic illustration of ligation reaction between the green fluorescent protein sfGFP engineered with a C-terminal tripeptide “NGL” 2 and the red fluorescent protein GI-mCherry 10. (B) Fluorescent gel analysis of the ligation reaction between sfGFP-NGL 2 and GI-mCherry 10 catalyzed by VyPAL2. Aliquots of the reaction mixture were taken out and frozen at different time points, and, at the end, subjected to SDS-page gel electrophoresis. (C) ESI-MS characterization of GI-mCherry 10, inter-protein ligation product 11, and the cyclic by-product 12 (10: calcd mass 28,860.4 and obsvd mass 28867.1; 11: calcd mass 56,350.5 and obsvd mass 56,354.1; 12: calcd mass 27,474.9 and obsvd mass 27,467.1). (D) Schematic illustration of the ligation reaction between the EGFR-targeting affibody Z_EGFR tagged with the C-terminal tripeptide “NGL” 13 and the red fluorescent protein GI-mCherry 10. (E) HPLC and ESI-MS analysis of the ligation reaction between 13 and 10 catalyzed by VyPAL2. 13 and 10 were mixed at a 2:1 ratio. The reaction was kept in room temperature for 30 min, then subjected to the HPLC analysis. ESI-MS characterization of Z_EGFR-NGL 13 and the inter-protein ligation product 14 (13: calcd mass 8799.7 and obsvd mass 8802.5; 14: calcd mass 37,481.2 and obsvd mass 37,490.9).

Figure 4

VyPAL2-mediated protein macrocyclization.

Figure 5

VyPAL-mediated macrocyclization of an EGFR-targeting affibody 15. (A) Scheme of VyPAL-mediated Z_EGFR 15 cyclization; (B) characterization of VyPAL-mediated Z_EGFR 15 cyclization by HPLC and ESI-MS analysis. Z_EGFR 15: calcd mass 8826.8 and obsvd m/z [M+H]⁺ 8827.3; product 17: calcd mass 8638.8 and obsvd m/z [M+H]⁺ 8638.0.

Figure 6

Receptor competitive binding assay to evaluate cyclic affibody Z_EGFR 17 and DARPin 18 binding to the target receptors. (A) Schematic representation of the receptor competitive binding assay using the cyclic affibody as the example; (B) flow cytometry analysis of cyclic affibody 17 binding to the EGFR receptor; (C) flow cytometry analysis of cyclic DARPin 18 binding to EGFR receptor.

Figure 7

Labeling of cell surface-bound affibody 3 and DARPin 4. (A) Reaction scheme of labelling protein ligands which are pre-bound to cell surface receptors; (B) flow cytometry characterization of A431 cells with pre-bound Z_EGFR 3 on cell surface after incubation with 5 in the presence or absence of VyPAL2; (C) flow cytometry characterization of A549 cells with pre-bound DARPin 4 on cell surface after incubation with 5 in the presence or absence of VyPAL2.

Figure 8

A cell surface modification strategy based on consecutive oxime ligation and VML. (A) Schematic illustration of cell surface modification in three steps: (1) sodium periodate oxidation of sialic acid on cell-surface glycans (by 10 mM sodium periodate in PBS at 0 °C for 5 min); (2) imine formation (oxime conjugation) by treating A431 cells with 1 mM of the aminooxyl-peptide 22 to anchor the PAL substrate onto the cell surface. (3) VML was performed by treating the cells with 0.5 mM of GI-peptide or GI-protein for 30 min. (B) Schematic illustration of VML for the labeling of pre-installed cell-surface VyPAL2 substrate with the fluorescein-peptide 5 (upper panel) or mCherry 10 (low panel). (C) Flowcytometry analysis of cells after labeling with fluorescein-peptide 5 using the three-step scheme. Time course study of sodium periodate treatment indicated the efficiency of a 5-min treatment. (D) Fluorescent microscopy imaging analysis of cells labeled with fluorescein-peptide 5 and mCherry 10. Negative control was done by treating A431 cells with 5 or 10 in the absence of VyPAL2.