Materials - Identification of the receptor for cellular internalization of an anti-nucleic acid

Heparin (Hp, #H3149), dextran sulfate (a synthetic analogue of Hp: DS, #D7037), chondroitin sulfate A (CS-A: #C9819), dermatan sulfate (CS-B: #C3788), chondroitin sulfate C (CS-C: #27043), non-enzymatic cell dissociation solution (#031M0935), rabbit IgG (#I5006), TRITC-goat anti-rabbit IgG (#T6778), ρ-nitrophenyl phosphate (ρ-NPP: #N2765) were obtained from Sigma (St.Louis, MO, USA). Mouse anti-heparan sulfate (10E4: #H1890) was purchased from USBiological (Swampscott, MA, USA), Alexa Fluor 488-goat anti-mouse IgM (μ chain-specific: #A21042), Alexa Fluor 647-goat anti-rabbit IgG (#A21244), AP-Streptavidine (#434322) were purchased from Invitrogen (Grand Island, NY, USA).

Mouse anti-chondroitin sulfate (#ab11570) was purchased from Abcam. Alkaline phosphatase (AP) conjugated-goat anti-rabbit IgG (#31341) and Hoechst 33342 (#62249) was purchased from PIERCE. Mouse anti-caveolin-1 (7C8: #sc-53564) and TRITC conjugated-anti-mouse IgG (#sc-3796) was obtained from SantaCruz. VECTASHIELD®

Mounting medium (#H-1000) was purchased from Vector Laboratories. Polyclonal rabbit anti-3D8 scFv was produced by immunization in our laboratory. The HIV-Tat peptide (amino acids 48-60 plus a biotin, Biotin-GRKKRRQRRRPPQ) was synthesized from Peptron (Daejeon, Korea).

５ B. Cells and Cell cultures

Wild type Chinese hamster ovary (CHO)-K1 cells (#CCL-61) and pan-GAG-deficient pgsA-745 mutant CHO cell (#CRL-2242) were purchased from the American Type Culture Collection (Manassas, VA, USA). Wild type and pan-GAG-deficient pgsA-745 mutant CHO cells were cultured in Ham’s F-12 medium supplemented with 10% FBS, and antibiotics (100U/ml penicillin and100 μg/ml streptomycin). Human epithelial cervical carcinoma-derived HeLa cells were grown in Dulbecco’s modified Eagle’s medium with the same supplements as above. Cells were cultured in a humidified 5% CO2 incubator at 37^oC.

C. Purification of scFv proteins

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 between His6 tag and pA.pIg20-3D8scFv, pIg20△ pA-3D8 scFv, and pIg20△ pA-HW6 scFv were transformed into Escherichia coli BL21(DE3) pLysE cells (Novagen). The transformants were cultured at 37^oC in Luria-Bertani medium containing ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml) until the absorbance at 600nm

reached 0.8. Isopropyl 1-thio-β-D-galactopyranoside (0.5 mM) was added to culture medium to induce the expression of proteins. Cells were then cultured at 23^oC for 18 h with shaking at 140 rpm. The culture supernatant was collected by centrifugation at 8,000 rpm for 30 min at 4^oC and filtered through a 0.45 μm cellulose acetate filter (Sartourius Stedim biotech) to

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remove cellular debris. The filtered culture supernatant was loaded to IgG-Sepharose 6 Fast Flow column (GE Healthcare) or HiTrap Protein L-agarose column (GE Healthcare). The column was washed with PBST (Phosphate-buffered saline, pH 7.4 plus 0.1% Tween 20) and 5 mM of ammonium acetate (pH 5.0). Proteins were eluted from the column with 0.1 M of acetic acid (pH 3.4). The eluted proteins were concentrated by centrifugation at 5,000 rpm at 4^oC using Vivaspin 20 (molecular weight cut off 10,000 Da: Sartorius Stedim biotech), and then buffer was changed to PBS (pH 6.0). The concentration of each protein was determined based on the molar extinction coefficient at 280 nm, which were calculated from the amino acid sequence.

D. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Purified proteins were diluted in 5X sample buffer (60 mM Tris, pH 6.8, 50%

glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromophenol blue in H2O) and boiled for 10 min at 100^oC. Protein samples (~ 5 μg) were submitted to electrophoresis on 12%

polyacrylamide gels for approximately 2 h at 100 V. Proteins were visualized with Coomassie brilliant blue.

E. Competitive ELISA

For detection of binding of 3D8 scFv△ pA to soluble GAGs, microtiter plates were coated with 10 μg/ml of CS-A or Hp as antigen at 4^oC overnight and blocked with TBST

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(Tris-buffered saline, pH 7.4 plus 0.1% Tween 20) containing 3% (w/v) bovine serum albumin (BSA) for 2 h at room temperature (RT). Plates were then added with various concentrations (0 – 100 μg/ml) of Hp and 3D8 scFv△ pA(20 μg/ml) or various concentrations (0 – 100 μg/ml) of CS-A and 3D8 scFv△ pA (20 μg/ml), respectively. Then, plates were incubated for 1 h at RT. After washing with TBST three times, the bound 3D8 scFv△ pA was detected with anti-His6 tag antibody and then AP-goat anti-mouse IgG

antibody. After washing, ρ-NPP solution (1 mg/ml in 0.1 M glycine, 1 mM ZnCl2, and 0.1 mM MgCl2, pH 10.4) was added and absorbance at 405 nm was measured with a microplate reader (Molecular Devices).

F. Flow cytometry

For detection of the internalized 3D8 scFv△ pA in the presence of soluble GAGs, 10 μM of 3D8 scFv△ pA was pre-incubated with soluble GAGs (Hp, DS, A, B, and CS-C: 100 μg/ml) for 30 min at 37^oC. HeLa cells were detached with a non-enzymatic cell

dissociation solution from cell culture dish and 6 x 10⁵HeLa cells were treated with pre-mixture and incubated for 6 h at 37ôC. After incubation, cells were washed with PBS three times by centrifugation at 1,500 rpm for 3 min at 4ôC. After washing, cells were treated with trypsin-EDTA for 10 min at 37ôC. Cells were fixed with 4% (w/v) paraformaldehyde/ PBS for 20 min at RT. Then cells were permeabilized with permeabilization buffer (1% BSA, 0.1%

saponin, and 0.1% sodium azide in PBS) for 1 h at 4^oC. After PBS washing, cells were incubated with rabbit anti-3D8 scFv antibody (1: 200) diluted in permeabilization buffer for

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1 h at 4^oC. Cells were washed with PBS three times and incubated with Alexa Flour 647-conjugated anti-rabbit IgG (1: 400 diluted in permeabilization buffer) for 1 h at 4^oC. After washing with PBS three times, cells were resuspended in 4% (w/v) paraformaldehyde/ PBS and analyzed with flow cytometer (BD FACS Canto II).

For detection of 3D8 scFv internalization in wild type and pan-GAG-deficient pgsA-745 mutant CHO cells, cells were detached with a non-enzymatic cell dissociation solution from cell culture dish and 1 x 10⁶cells were incubated with 10 μM of 3D8 scFv for the indicated times at 37ôC. After incubation, cells were washed with PBS three times by centrifugation at 1,500 rpm for 3 min at 4ôC. After washing cells were treated with trypsin-EDTA for 10 min at 37ôC. Cells were fixed with 4% (w/v) paraformaldehyde/ PBS for 20 min at RT. Then cells were permeabilized with permeabilization buffer for 1 h at 4ôC. After PBS washing, cells were incubated with rabbit anti-3D8 scFv antibody (1: 200) diluted in soluble GAGs, HeLa cells were seeded in 24-well plate with cover slips at 4 x 10⁴cells/ well.

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3D8 scFv△ pA (10 μM) was pre-incubated with soluble GAGs (Hp and CS-A: 100 μg/ml)

for 30 min at 4^oC. The pre-mixture was treated to HeLa cells and incubated for 1 h at 4^oC.

Cells were washed with PBS five times in shaker and then fixed in 4% (w/v) paraformaldehyde for 10 min at RT. After three times washing with PBS, cells were incubated with the mixture of rabbit anti-3D8 scFv antibody (1: 200) and mouse anti-heparan sulfate antibody (1: 200) diluted in the surface buffer (0.5% BSA and 2 mM EDTA in PBS, pH 8.5) at 4^oC overnight. Cells were then washed with PBS five times and incubated with the mixture of TRITC-conjugated anti-rabbit IgG (1: 200) and Alexa Fluor 488-conjugated anti-mouse IgM (μ chain-specific: 1: 200) diluted in the surface buffer for 1 h at RT. After five times washing with PBS, nuclei were stained with Hoechst 33342 (20 μg/ml).After washing with PBS once, cells were mounted in Vectashield, and then analyzed using Zeiss LSM 710 laser confocal microscope equipment with a 40 X 1.2 NA water immersion objective.

For detection of interaction of 3D8 scFv△ pA and cell surface PGs, HeLa cells were incubated with 10 μM of 3D8 scFv△ pA for 1 h at 4^oC. Cells were washed with PBS five

times in shaker and then fixed in 4% (w/v) paraformaldihyde for 10 min at RT. After three times washing with PBS, cells were incubated with either the mixture of rabbit anti-3D8 scFv antibody (1: 200) and mouse anti-heparan sulfate antibody (1: 200), or the mixture of rabbit anti-3D8 scFv antibody (1: 200) and mouse anti-chondroitin sulfate antibody (1: 200) diluted in the surface buffer at 4^oC overnight. After washing with PBS five times and cells were incubated with the mixture of TRITC-conjugated anti-rabbit IgG (1: 200) and Alexa

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Fluor 488-conjugated anti-mouse IgM (μ chain-specific: 1: 200) diluted in the surface buffer for 1 h at RT. After five times washing with PBS, nuclei were stained with Hoechst 33342.

After washing with PBS once, cells were mounted in Vectashield, and then analyzed using Zeiss LSM 710 laser confocal microscope equipment with a 40 X 1.2 NA water immersion objective.

For examination of intracellular co-localization, HeLa cells were incubated with 10 μM of 3D8 scFv△ pA for the indicated times at 37^oC. Following incubation, cells were fixed

with 4% (w/v) paraformaldehyde/ PBS for 10 min at RT and permeabilized with permeabilization buffer for 10 min at RT. After washing with PBS three times, cells were incubated with either the mixture of rabbit anti-3D8 scFv (1: 200), mouse anti-caveolin 1 antibody (1: 200) and mouse anti-heparan sulfate antibody (1: 100), or the mixture of rabbit anti-3D8 scFv (1: 200), mouse anti-caveolin 1 antibody (1: 200), and mouse anti-chondroitin sulfate antibody (1: 100) diluted in permeabilization buffer for overnight at 4^oC. After washing with PBS five times, cells were incubated with the mixture of Alexa Flour 647-conjugated anti-rabbit IgG (1: 200), TRITC-647-conjugated anti-mouse IgG (1: 200), and Alexa Flour 488-conjugated anti-mouse IgM (μ chain-specific) (1: 200) diluted in permeabilization buffer for 1 h at RT. After five times washing with PBS, cells were mounted in Vectashield, and then analyzed using Zeiss LSM 710 laser confocal microscope equipment with a 40 X 1.2 NA water immersion objective.

For detection of the internalized 3D8 scFv in wild type and pan-GAG-deficient pgsA-745 mutant CHO cells, cells were seeded in 24-well plate with cover slips at 4 x 10⁴

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cells/ well. After 1 day, cells were incubated with 10 μM of 3D8 scFv for 2 h at 37^oC. After incubation, cells were washed with PBS five times in shaker. Cells were then fixed and permeabilized. After PBS washing, cells were incubated with rabbit IgG (10 μg/ml) diluted in permeabilization buffer for 1 h at RT. Cells were washed with PBS five times and incubated with FITC-conjugated anti-rabbit IgG (1: 200 diluted in permeabilization buffer) for 1 h at RT. After five times washing with PBS, nuclei were stained with Hoechst 33342.

After washing with PBS once, cells were mounted in Vectashield, and then analyzed using Zeiss LSM 710 laser confocal microscope equipment with a 40 X 1.2 NA water immersion objective.

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III. RESULTS

A. Purify the 3D8 and HW6 proteins.

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 37^oC. HeLa cells were incubated with the pre-mixture for 6 h at 37^oC.

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 37^oC.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 37^oC.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 4^oC.

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 4^oC. 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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D. Internalized 3D8 scFv△ pA co-localizes with HSPGs, CSPGs, and caveolin 1.

Previous our group has demonstrated that 3D8 scFv internalization occurs by caveolae/lipid-raft-mediated endocytosis in HeLa cells (Jang et al., 2009). Accordingly, we speculated that the internalized 3D8 scFv△ pA with either HSPGs and CSPGs would co-localize with caveolin1, caveolar structural protein, in cytosol. HeLa cells were incubated with 3D8 scFv△ pA and then we detected the internalized 3D8 scFv△ pA, PGs, and caveolin1 by confocal microscopy. Intracellular co-localization of 3D8 scFv△ pA and HSPGs was detected on 6 h and 12 h incubation, but not on 2 h (Fig.6A). Intracellular co-localization of 3D8 scFv△ pA and CSPGs was observed on 2 h, 6 h, and 12 h incubation (Fig.

6B). Representative images show the intracellular co-localization of 3D8 scFv△ pA-PGs (yellow), 3D8 scFv△ caveolin 1 (magenta), PGs-caveolin1 (cyan), or 3D8 scFv△ pA-PGs-caveolin 1 (white) during endocytosis. These results show that internalization of 3D8 scFv△ pA is mediated by cell surface HSPGs and CSPGs. Besides, this result is consistent with previous data that 3D8 scFv internalizes through caveolae/lipid-raft-mediated endocytosis.

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Figure 6. Intracellular co-localization between 3D8 scFv△ pA, HSPGs, CSPGs, and caveolin 1. HeLa cells were treated with 10 μM of 3D8 scFv△ pA for 2 h, 6 h, and 12 h at

37^oC. After fixation and permeabilization of cell membrane, cells were incubated with the mixture of three antibodies of Alexa Flour 488-conjugated anti-mouse IgM, Alexa Flour 647-conjugated anti-rabbit IgG, and TRITC-647-conjugated anti-mouse IgG. The co-localization of 3D8 scFv△ pA-PGs (yellow), 3D8 scFv△ pA-caveolin 1 (magenta), PGs-caveolin 1 (cyan), or 3D8 scFv△ pA-PGs-caveolin 1 (white) was detected by confocal microscopy. Under Detail, the enlarged images of the indicated region show co-localization in detail. Bar, 10 μm or 5 μm (Detail panels).

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IV. DISCUSSION

A subset of monoclonal anti-DNA antibodies can internalize cells and then translocate the nucleus in variety of cells (Yanase et al., 1994; Zack et al., 1996; Ternynck et al., 1998; Lee et al., 2007). Up to date, function of HSPGs and CSPGs as an endocytic receptor for the internalization of anti-DNA Abs has not been studied. Here, we report first that cell surface HSPGs and CSPGs are true internalizing receptor for anti-nucleic acid antibody, 3D8 scFv.

So far several membrane components, such as calreticulin, myosin 1, and equilibrative nucleoside salvage transporter (ENT), have been suggested as potential cell surface receptors for the internalization of anti-DNA Abs. Anti-dsDNA mAb was also reported not to require a cell-membrane receptor since cell-penetration was not inhibited by endocytosis inhibitors (Song et al., 2008). It was from the findings that calreticulin (for mAbs F14.6 and H9.3) (Seddiki et al., 2001) or myosin 1 (for mAb H7) (Yanase et al., 1997) has been immunocaptured from the cells treated with the anti-DNA Abs, and 3E10 mAb was unable to penetrate into ENT-deficient cells (Hansen et al., 2007). Moreover, 9D7 anti-dsDNA mAb was reported not to require a cell-membrane receptor since cell-penetration was not inhibited by endocytosis inhibitors (Song et al., 2008). Therefore, it seems that different anti-DNA Abs may preferentially take distinct pathways of penetration.

Human and murine anti-DNA Abs are cross-reactive with HSPGs, the major GAG in glomerular basement membranes (Faaber et al., 1986; Naparstek et al., 1990). Mice

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immunization with HS induced systemic lupus erythematosus-like disease (Ofosu-Appiah et al., 1998). Hp-affinity chromatography has been widely used for the purification of DNA-binding proteins, such as transcription factors, steroid hormone receptors, and histones (Belting, 2003). Despite accumulated documents for cross-reactivity of DNA-binding proteins to HS, the involvement of cell surface HSPGs in the uptake of anti-DNA Abs have not been demonstrated before our study. Moreover, previous our work showed that 3D8 scFv internalization was inhibited in the presence of soluble Hp in HeLa cells (Jang et al., 2009).

Based on these studies, we hypothesized that 3D8 scFv, a basic protein (pI = 9.15), enters the cells by interacting with cell surface HSPGs and CSPGs which are negatively charged molecules on the plasma membrane. Our result showed that 3D8 scFv△ pA internalization was inhibited in the presence of soluble CS as well as Hp (Fig. 2) and 3D8 scFv△ pA bound to cell surface CSPGs as well as HSPGs to a similar extent (Fig. 4). From these data, it is clear that 3D8 scFv△ pA interacts with both cell surface CSPGs as well as HSPGs without

any preference before it penetrates into cells.

HSPGs have been proposed to act either as true internalizing receptors or as co-receptors for temporary cell surface attachment in the internalization of a variety of macromolecules because it has been shown that the internalization of macromolecules such as DNA, cationic polymers, and cell-penetrating peptides is dependent on the presence of the intact cell surface HSPGs. Recently a direct evidence for role as a true internalizing receptors of HSPGs was provided by the observation of translocation of intact cell surface HSPGs (syndecan and glypican) to endocytic vesicles induced by anti-HS scFv (AO4B08), depending on the interaction with HS 2-O-sulfation (Wittrup et al., 2009). Like anti-HS scFv,

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3D8 scFv internalized along with HSPGs, supported by the observation of co-localization of 3D8 scFv with HSPGs on cell surface and inside cells during endocytosis (Fig. 4C and 6A).

Compared to numerous documents for endocytosis via binding of macromolecules

upon HSPGs, only a few is known for involvement of cell surface CSPGs in endocytosis.

Low density lipoprotein (LDL) interacts with CSPGs, internalized, and degraded in human macrophage (Hurt-Camejo et al., 1990). Yang et al have shown that intracellular translocation and cytotoxicity of penetratin-directed mitochondria-disrupting peptides was reduced by the treatment of soluble CS or chondroitinase, and was positively correlated to expression level of CS on cell surface. They suggested that CS overexpression in tumor cells

문서에서 Identification of the receptor for cellular internalization of an anti-nucleic acid antibody, 3D8 scFv (페이지 12-0)