pIg20 vector was used to express scFv proteins with both His6 tag and Staphyplococcal protein A tag (pA). pIg20△ pA was constructed to express scFv without pA
by inserting a stop codon upstream of pA (Fig. 1A). 3D8 and HW6 proteins were produced from bacterial expression system. The purity of purified proteins was ~90% on SDS-PAGE gels (Fig. 1B). The yields of 3D8 scFv, 3D8 scFv△ pA, and HW6 scFv△ pA were about 4-6 mg/L, 1-2 mg/L, and 2-3 mg/L, respectively.
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Figure 1. Production of scFv proteins. (A) Schematic diagram showing a part of the expression vectors for the indicated proteins. (B) SDS-PAGE of purified proteins. The plasmids encoding the 3D8 scFv (~ 34 kDa), 3D8 scFv△ pA (~ 27 kDa), and HW6 scFv△ pA (~ 28 kDa) were transformed into E.coli and purified from the culture supernatant
using IgG-Sepharose column or Protein L-agarose column. Proteins (5μg) were run on a 12%
polyacrylamide gel and visualized with Coomassie brilliant blue.
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B. 3D8 internalization depends on HSPGs and CSPGs.
Previous works by another groups have reported that cell-penetrating peptide and cationic lipids enter the cells by interacting with HSPGs (Goncalves et al., 2005; Poon and Gariepy, 2007; Song et al., 2008). Moreover, previous our work has shown that 3D8 scFv internalization was reduced in the presence of soluble Hp that is a high-sulfated HS (Dreyfuss et al., 2009) in HeLa cells (Jang et al., 2009). Here, we examined whether anionic cell surface CSPGs as well as HSPGs can act as internalizing sites for 3D8 scFv. When HeLa cells were incubated with 3D8 scFv△ pA in the presence of soluble GAG, internalization of 3D8 scFv△ pA was significantly inhibited (Fig. 2). Our results suggest that cell surface
HSPGs and CSPGs are required for 3D8 scFv internalization.
Next, we carried out flow cytometry with wild type and pan-GAG-deficient pgsA-745 mutant CHO cells. When wild type and pan-GAG-deficient pgsA-pgsA-745 mutant CHO cells were incubated with 3D8 scFv, internalization of 3D8 scFv was severely impaired in pan-GAG-deficient pgsA-745 mutant CHO cells compared with that in wild type CHO cells (Fig.
3A). This data was consistent with that by confocal microscopy (Fig. 3B). These results support the data shown in Fig. 2.
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Figure 2. Effects of soluble GAGs on 3D8 scFv△ pA internalization. 3D8 scFv△ pA (10 μM) was pre-incubated with soluble GAGs such as Hp, DS, CS-A, CS-B, or CS-C (100 μg/ml) for 30 min at 37oC. HeLa cells were incubated with the pre-mixture for 6 h at 37oC.
After fixation and permeabilization of cell membrane, the internalized 3D8 scFv△ pA was detected with rabbit anti-3D8 scFv antibody and Alexa Flour 647-conjugated anti-rabbit IgG, prior to flow cytometry.
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Figure 3. 3D8 scFvinternaization in wild type and pan-GAG deficient pgsA-745 mutant CHO cells. (A) Wild type and pan-GAG deficient pgsA-745 mutant CHO cells were incubated with 3D8 scFv (10 μM) for the indicated time at 37oC.After fixation and permeabilization of cell membrane, the internalized 3D8 scFv was detected with rabbit anti-3D8 scFv antibody and Alexa Fluor 647-conjugated anti-rabbit IgG, prior to flow cytometry.
(B) Wild type and pan-GAG deficient pgsA-745 mutant CHO cells were incubated with 3D8 scFv (10 μM) for 2 h at 37oC.After fixation and permeabilization of cell membrane, the internalized 3D8 scFv (green) was detected with rabbit IgG and FITC-conjugated anti-rabbit IgG, prior to confocal microscopy. Bar, 10 μm.
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C. 3D8 scFv△ pA binds to cell surface HSPGs and CSPGs.
To confirm the binding of 3D8 scFv△ pA to cell surface HSPGs and CSPGs, we performed competitive ELISA. 3D8 scFv△ pA was pre-incubated with Hp as a competitor and then this pre-mixture was added to CS-A-coated plate. The binding of 3D8 scFv△ pA to CS-A was decreased in an Hp concentration-dependent manner (Fig. 4A). In reverse, when we used Hp as an antigen and CS-A as a competitor, binding of 3D8 scFv△ pA to Hp was decreased in a CS-A concentration-dependent manner (Fig. 4B). In confocal microscopy, co-localization of 3D8 scFv△ pA, cell surface HSPGs, and CSPGs was observed on the surface of HeLa cells treated with 3D8 scFv△ pA (Fig. 4C and D).
To investigate whether HSPGs compete with CSPGs for 3D8scFv△ pA-binding on the cell surface, we incubated HeLa cells with 3D8 scFv△ pA in the presence of soluble Hp or CS-A. Binding of 3D8 scFv△ pA to cell surface was markedly reduced in the presence of soluble Hp or CS-A (Fig. 5). Overall, these results show that 3D8 scFv△ pA bind to both of cell surface HSPGs and CSPGs on HeLa cells.
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Figure 4. Binding of 3D8 scFv△ pA to HSPGs and CSPGs. (A) Competitive ELISA. CS-A (10 μg/ml) was coated on ELISCS-A plate. 3D8 scFv△ pCS-A (20 μg/ml) was pre-incubated with Hp (0 - 100 μg/ml) for 30 min at RT. The 3D8 scFv△ pA bound to CS-A was detected with mouse anti-His6 tag antibody, and then AP-conjugated anti-mouse IgG. (B) Competitive ELISA. Hp (10 μg/ml) was coated on plate. 3D8 scFv△ pA (20 μg/ml) was pre-incubated with CS-A (0 - 100 μg/ml) for 30 min at RT. The 3D8 scFv△ pA bound to Hp was detected
with mouse anti-His6 tag antibody, and then AP-conjugated anti-mouse IgG. Experiments were done in triplicate. (C and D) Confocal microscopy. HeLa cells were incubated with HW6 scFv△ pA (10 μM) as negative control and 3D8 scFv△ pA (10 μM) for 1 h at 4oC.
Then, cells were incubated with the mixture of Alexa Flour 488-conjugated anti-mouse IgM and TRITC-conjugated anti-rabbit IgG. Binding of scFv△ pA proteins (red) to cell surface HSPGs (C, green) or CSPGs (D, green) was visualized by confocal microscopy. Bar, 10 μm.
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Figure 5. Binding of 3D8 scFv△ pA to cell surface HSPGs in the presence of soluble GAGs. HeLa cells were incubated with 3D8 scFv△ pA (10 μM) in the absence (upper panel)
or in the presence (middle or lower panel) of Hp (100 μg/ml) or CS-A (100 μg/ml) for 1 h at 4oC. Then, cells were incubated with the mixture of Alexa Flour 488-conjugated anti-mouse IgM and TRITC-conjugated anti-rabbit IgG. The 3D8 scFv△ pA (red) bound to cell surface was detected by confocal microscopy. Bar, 10 μm.
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