Proteins, antibodies, and cell culture

The recombinant proteins used in this study were provided by Protgen Co., Ltd. (Beijing, China). ES and ZBP-ES were expressed in E. coli, refolded into native forms and purified. The N-terminal site-directed mono-PEGylation was conducted by addition of 20 mmol/L NaBH₃CN and incubating the PEG2000 and proteins (PEG2000:protein = 1.5:1) at room temperature for more than 2 h, followed by purification.

We purchased primary antibodies against extracellular regulated protein kinases (Erk), phosphor-Erk, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PEG (Abcam, Cambridge, UK), and turbo green fluorescent protein (tGFP) (OriGene, Rockville, MD, USA). Anti-ES antibody was from our lab stock.

Human microvascular endothelial cells (HMECs) were from our lab stock and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA).

Circular dichroism (CD)

The far-ultraviolet (UV) CD spectra were obtained by a Chirascan™-plus CD Spectrometer (Applied photophysics, Surrey, UK). ES or its variants were diluted in 5 mmol/L Tris–HCl, pH 7.4, to a final concentration of 10 μmol/L. For each protein, data were recorded three times and corrected by subtracting the baseline spectrum of the buffer.

Tryptophan emission fluorescence

Tryptophan emission fluorescence is a good probe to monitor the subtle tertiary structural changes of ES [16]. Proteins (1 μmol/L) in Tris buffer were measured as previously described [17].

Proteolysis assay

ES or its variants (0.5 mg/mL) were incubated with trypsin (0.2 mg/mL) in phosphate buffer saline (PBS) buffer (pH 7.4) at 37 °C [18]. At each indicated time point, reaction solutions were quickly removed and mixed with the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer to stop the trypsin digestion reactions. The samples were subsequently subjected to SDS–PAGE and stained with the Coomassie dye.

Guanidinium chloride (GdmCl)-induced unfolding

ES or its variants (0.8 μmol/L) were incubated in 5 mmol/L Tris–HCl, pH 7.4, containing GdmCl concentrations ranging from 0 to 6 mol/L. After incubation for 24 h at room temperature, GdmCl-induced denaturation was monitored by the tryptophan emission fluorescence intensity at 318 nm [5, 19]. Data were normalized by subtracting the baseline fluorescence intensity of the buffer. The linear extrapolation method was used to evaluate the value of the free change energy in the absence of the denaturant GdmCl (\(\rm\Delta {\text{G}}^{\text{o}}_{{{\text{N}}{-}{\text{U}}}}\)), the apparent slope of the plots (m), and the concentration of GdmCl at the midpoint of the unfolding transition (C_m) as described by Santoro and Bolen [20].

In vitro protein–protein interactions

The nucleolin-tGFP plasmid was transfected into HMECs. Nucleolin-tGFP proteins were immunoprecipitated following the procedure described in our previous study [21]. Subsequently, ES or its variants were incubated with the nucleolin-tGFP beads for 1 h at 4 °C. After washing with the lysis buffer (150 mmol/L Tris, 50 mmol/L sodium chloride, 1% NP40, protease inhibitor mixture, pH 7.4), the bead-bound proteins were subjected to Western blotting.

Internalization assay

As previously described [22], HMECs were treated with 5 μg/mL ES or its variants for 1 h. Subsequently, cells were washed with acidic buffer (pH 3.5) and ice-cold PBS to remove cell surface-binding proteins. The amount of internalized proteins was examined by Western blotting or immunofluorescence.

Western blotting

Protein samples were separated by SDS–PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking for 1 h at room temperature, the membrane was incubated with the indicated primary antibodies overnight and then to the corresponding horseradish peroxidase (HRP)-conjugated goat-anti-mouse or goat-anti-rabbit secondary antibodies for 1 h. The enhanced chemiluminescent substrate was added to the blot, and reactive bands were detected.

Immunofluorescence

Cells were seeded directly on coverslips, fixed, and stained. After blocking with 5% goat serum, cells were incubated with the indicated primary antibodies and fluorescein isothiocyanate (FITC)-labeled secondary antibodies. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Nikon A1 confocal microscope (Nikon Corporation, Tokyo, Japan).

Transwell migration and wound healing

Transwell migration and wound healing assays were conducted as previously described [22]. Cells were treated with the indicated proteins for 6 and 48 h at 37 °C, respectively. All experiments were repeated twice.

Statistical analyses

All data from the experiments were presented as means ± standard deviations (SDs). Differences between two groups were calculated by GraphPad (GraphPad Software, San Diego, CA, USA), and considered significant if the P value was <0.05 using a two-tailed Student’s t test.

Structural analyses

The schematic diagram shows the sequence of ES, ZBP-ES, M₂ES, and MZBP-ES (Fig. 1a). The purity of these proteins is shown in Fig. 1b. The proteins were tested for secondary and tertiary structures using CD and tryptophan emission fluorescence. The CD spectra results revealed that no obvious change in the secondary structure was observed between ES and ZBP-ES, whereas slight differences were observed between M₂ES and MZBP-ES (Fig. 1c). The maximal Trp fluorescence emission wavelengths (λ _max) of all the ES variants were approximately 318 nm (Fig. 1d), indicating a relatively stable core structure of ES variants. However, the fluorescence intensities of ES and M₂ES were higher than those of ZBP-ES and MZBP-ES, respectively, implying that the tertiary structure of ES is more compact than that of ZBP-ES and that the tertiary structure of M₂ES is more compact than that of MZBP-ES.

Taken together, N-terminal modifications do not affect the core structure of ES, whereas the tertiary structure of M₂ES is more compact than that of MZBP-ES.

Protease digestion

To identify the stability of ES variants, trypsin was incubated with ES variants for the indicated time periods. ES was quickly digested within 20 min, whereas ZBP-ES was not totally digested until 4 h (Fig. 2a). PEGylated proteins were overall more resistant to trypsin digestion compared with intact proteins. Interestingly, after incubation for 6 h, the residual amount of full-length M₂ES was larger than that of MZBP-ES. Quantitative results showed that the initial reaction rate was 0.026 mg/(mL·min) for M₂ES and 0.037 mg/(mL·min) for MZBP-ES. These results showed that M₂ES was more resistant to trypsin digestion than MZBP-ES.

GdmCl-induced unfolding

Biological activities

We next investigated the effects of ES variants on endothelial cell activities. ES and its variants significantly reduced endothelial cell migration, and PEGylated proteins were more efficient compared with intact proteins (Fig. 3a, b). In the Transwell assay, M₂ES and MZBP-ES inhibited endothelial cell migration to an equivalent extent. The wound healing scratch results showed that M₂ES was slightly more potent than MZBP-ES in retarding wound healing (Fig. 3c).

ES was shown to bind to the vascular endothelial growth factor receptor 2 (VEGFR2) on the surface of endothelial cells and disrupt vascular endothelial growth factor (VEGF)-induced activation of mitogen-activated protein kinase/extracellular regulated protein kinases (MAPK/Erk) [23]. All ES variants inhibited Erk phosphorylation (Fig. 3d). ZBP-ES was more potent in reducing Erk activation compared with ES, whereas the blockage effect of M₂ES was more potent than that of MZBP-ES.

In summary, M₂ES and MZBP-ES had comparable effects on the inhibition of cell migration, whereas M₂ES was more efficient in disrupting Erk activation.

Internalization by endothelial cells and interactions with cell surface receptors

Internalization is important, if not critical, for the biological functions of ES [24]. Increasing the uptake of ES via the addition of cholesterol chelating agents [22] or engineering a peptide to its N terminus [25] dramatically enhanced the therapeutic efficacy of ES. We determined the uptake of ES and its variants by HMECs and found that ZBP-ES was more efficient in entering the cells than ES (Fig. 4a), which validates the data from our previous study [16]. Intriguingly, more M₂ES proteins accumulated in the cells than MZBP-ES (Fig. 4a). Consistent results were obtained by immunofluorescence (Fig. 4b).

Nucleolin is a functional receptor of ES and participates in the ES internalization process [26]; therefore, we explored whether the distinction of ES variant internalization was attributed to different binding affinities to nucleolin. Consistent with the internalization results (Fig. 4a, b), ES showed weaker interaction with nucleolin than ZBP-ES, whereas M₂ES showed increased interaction with nucleolin compared with MZBP-ES (Fig. 4c).

Collectively, M₂ES exhibited an increased cellular uptake than MZBP-ES, possibly caused by the enhanced interaction with its functional receptor nucleolin.