Comparisons of biophysical properties and bioactivities of mono-PEGylated endostatin and an endostatin analog

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 NaBH
₃
CN 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 Chirascanâ„¢-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 ?mol/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 ?mol/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 ?mol/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 (), 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 ?g/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.