Expression of IgGs in the cytosol - Structural and Functional Properties of the Recombinant ant

To investigate whether the characteristics of the antibody expressed in the cytosol are determined according to the variable region sequence, chimeric IgG was constructed by fusing four variable regions of mouse-derived antibodies (3 anti-KIFC1 antibodies and an anti-nucleic acid antibody) with the human IgG1 constant region (Figure 4A). A heavy chain and a light chain gene of IgG were inserted into a vector containing two promoters so that each chain could be expressed simultaneously. At the N-terminus of each chain, there is a leader sequence that transfers proteins to the ER when protein translation begins. The IgG expressed in ER was designated Ld. Cytosolic expression was achieved by removing the leader sequence at the N-terminus of the heavy and light chains, and the antibody expressed in the cytosol was labeled as ΔLd. IgGs were expressed in the ER or cytosol of HEK293T cells and expression level was confirmed by immunoblotting (Figure 4B). In the results, all heavy chains were expressed at similar levels regardless of the leader sequence. Light chain of 3D8 and 10C358 showed slightly lower cytosolic expression levels than those expressed in ER (Figure 4B).

Figure 4. Expression of IgGs in the ER or cytosol of HEK293T cells. (A) Schematic diagram of plasmids construction to express chimeric IgGs in the ER (Ld) or cytosol (ΔLd).

Heavy and light chains of IgG were expressed simultaneously using dual promoter vector.

(B) Immunoblotting for detection of heavy and light chains in lysates of HEK293T cells. An anti-DNA antibody (3D8) and three anti-KIFC1 antibodies (2C281, 6C407, and 10C358) were expressed in the ER or cytosol of HEK293T cells. The expressions of heavy and light chains were detected by goat anti-human IgG (Fc specific) antibody and goat anti-human kappa chain antibody followed by HRP-conjugated rabbit anti-goat IgG (H+L).

29 B. Antigen-binding activity of cytosolic IgGs

Antigen-binding activity of cytosolic IgGs was confirmed by ELISA and confocal microscopy. In ELISA, cell lysates prepared by disrupting HEK293T cells expressing cytosolic IgG were used. The ELISA plate coated with KIFC1 peptide antigens for anti-KIFC1 antibodies or single-strand DNA antigen for 3D8. All antibodies, except 10C358, expressed in the cytosol showed antigen-binding activity. All the antibodies expressed in ER had antigen-binding activity (Figure 5). This result was confirmed again by confocal microscopy (Figure 6). To confirm whether the cytosolic anti-KIFC1 IgG binds not only to the peptide antigen but also to the intracellular full-size KIFC1, co-localization of cytosolic anti-KIFC1 IgG with GFP-KIFC1 expressed in the HeLa was observed by confocal microscopy (Figure 6A). In interphase, KIFC1 is in the nucleus and cannot interact with IgG expressed in the cytosol. KIFC1 binds to the microtubule that forms a mitotic spindle in the metaphase of mitosis and moves toward the centrosome (minus-directed). Therefore, anti-KIFC1 IgG expressed in the cytosol can bind to anti-KIFC1 in mitosis. Similar to the cell lysate ELISA results, of the three anti-KIFC1 IgGs, 6C407 and 2C281 co-localized with KIFC1 in the mitotic phase, whereas 10C358 did not (Figure 6A). In the case of cytosolic 3D8 IgG, there is no antigen that can be used in cells. To confirm whether the antigen-binding site of cytosolic 3D8 IgG was properly formed, O2F3 IgM, an anti-idiotypic antibody that recognizes the conformation of the variable region of 3D8 IgG, were used to confocal microscopy (Figure 6B). HEK293T cells expressing cytosolic 3D8 IgG were fixed and permeabilized, and then treated with O2F3 IgM and its specific fluorescent-labeled antibody.

Confocal microscopy confirmed that cytosolic 3D8 IgG was recognized by O2F3, indicating

that the antigen binding site of cytosolic 3D8 IgG is properly formed. The results of the antigen-binding activity of cytosolic IgG showed that the variable region of the antibody determines the antigen-binding activity of cytosolic IgG.

Figure 5. Evaluation of binding activity of cytosolic IgGs. (A) The antigen-binding activity of cytosolic anti-KIFC1 IgG was analyzed by ELISA using peptide antigens synthesizing the epitope amino acid sequence of each antibody (KIFC1 peptide #1 for 2C281,

#2 for 6C407, and #3 for 10C358). Lysates of transfectants were placed in wells coated with specific antigens and bound IgGs were detected with AP-conjugated anti-human IgG/Fc.

Purified IgGs from HEK293F cells were used as positive control. (B) Binding activity of 3D8 anti-DNA IgG was analyzed by ELISA using synthetic single-strand DNA antigen. The subsequent ELISA procedure was performed in the same manner as in (A). Data are presented as mean ± SEM, n = 3. (C) An illustration showing the progress of the experiment.

Figure 6. Confocal microscopic analysis to confirm antigen-binding site of the cytosolic IgGs. (A) Cellular antigen-binding activity of cytosolic anti-KIFC1 IgGs were analyzed by confocal microscopy. HeLa cells stably expressing GFP-KIFC1 were transfected with the plasmids encoding ΔLd-anti-KIFC1 IgG gene. After synchronization of cells to mitotic phase, cells were fixed and stained with a primary antibody for anti-human IgG/Fc, followed by rhodamine-conjugated anti-goat IgG. (B) Formation of antigen-binding site of cytosolic 3D8 IgG was analyzed by confocal microscopy. Transiently transfected HEK293T cells were fixed, permeabilized, and then incubated with O2F3 (mouse IgM), followed by an Alexa Fluor 647-conjugated anti-mouse IgM/μ chain antibody. Bar = 10 μm.

C. Association of heavy and light chains of cytosolic IgGs

To explore why the antigen-binding activities of cytosolic IgGs are different each other, association between heavy and light chains of IgG was examined. We examined the association between heavy and light chains that compose the antigen binding site. Heavy chains of IgG in the HEK293T transfectants were immuno-precipitated by Protein-A/G and light chains interacting with the heavy chains were detected by western blot (Figure 7). As a result, H:L association was not observed in cytosolic 10C358 IgG that lost antigen-binding activity when expressed in the cytosol. The association of heavy and light chains of IgG was also confirmed through ELISA (Figure 8). Heavy chain of cytosolic IgG in the cell lysate is captured by Fc-specific antibody that is coated on the ELISA plate. The light chain associated with the heavy chain of cytosolic IgG is detected as a Cκ-specific antibody, and the AP-conjugated secondary antibody and the substrate are treated sequentially. ELISA was also performed to reverse the order of the antibodies used for capture and detection. After coating the Ck-specific antibody on the plate, the light chain of the cytosolic IgG was captured, and the heavy chain associated with the light chain was detected as AP-conjugated Fc-specific antibody. As a result, association between heavy and light chains was not observed in cytosolic 10C358 IgG as in IP results. These results confirm that the difference in antigen-binding of cytosolic IgG is related to the association of heavy and light chains.

Figure 7. Association of heavy and light chains of cytosolic IgGs. HEK293T cells lysates of IgGs transfectants were immunoprecipitated using Protein A/G-agarose. Input and IP samples were resolved by reducing SDS-PAGE and detected with goat anti-human IgG (Fc specific), goat anti-human IgG (kappa chain specific), and rabbit anti-cytoskeletal actin antibody, followed by HRP-conjugated rabbit goat IgG and HRP-conjugated goat anti-rabbit IgG, respectively. Input represents 10% of the total amount of lysate used in for IP.

Figure 8. Evaluation of H:L association of cytosolic IgGs. (A) Anti-human IgG/Fc was used as a capture antibody, followed by treatment of IgGs transfectants. Bound IgGs were detected by anti-human C and AP-conjugated secondary antibody. Asterisk (*) indicates negative control to ensure that the anti-rabbit IgG/Fc-specific antibody does not directly react with human IgG/Fc. (B) Anti-human C was used as a capture antibody. Lysates of HEK293T cells expressing IgGs were treated and detected by AP-conjugated anti-human IgG/Fc. Data are presented as mean ± SEM, n = 3.

D. H:L association in the absence of formation of the correct antigen-binding site If the antigen-binding activity of the variable region is not present, does it mean that the H:L association not occur? To explore this, hybrid 2C281 was constructed by replacing the Vκ site of cytosolic 2C281 IgG with a pseudo Vκ that does not bind to KIFC1, thereby eliminating the antigen-binding activity and comparing its characteristics with the original 2C281 IgG (Figure 9A). ELISA was performed to investigate antigen-binding activity and confirmed that the cytosolic hybrid 2C281 IgG did not bind to its peptide antigen (Figure 9B).

However, the results of H:L assembly-ELISA and IP showed that the heavy and light chain assembly of the cytosolic hybrid 2C281 IgG was maintained (Figure 9C, D). It was confirmed that even though the antigen-binding site is not properly formed, H:L association can occur irrespective of the antigen-binding site formation.

Figure 9. Association of heavy and light chains without formation of the correct antigen-binding site. (A) Schematic representation of Ld-hybrid IgG1 possessing 2C281 VH and pseudo Vκ as variable regions. (B) Evaluation of antigen-binding activity by ELISA. Lysates of HEK293T cells expressing ΔLd-2C281 or ΔLd-hybrid IgG were treated in wells coated with KIFC1 #1 peptide and bound chimeric IgGs were detected with AP-conjugated anti-human IgG/Fc antibody. (C) Evaluation of association between heavy and light chains by ELISA. Lysates of HEK293T cells expressing ΔLd-2C281 or ΔLd-hybrid IgG were treated in wells coated with anti-human C, and bound IgGs were detected with AP-conjugated anti-human IgG/Fc antibody. In (B, C), data are presented as mean ± SEM, n = 6. (D) Co-IP to

confirm assembly of heavy and light chains. Lysates of HEK293T cells expressing ΔLd-2C281 or ΔLd-hybrid IgG were immunoprecipitated using Protein A/G-agarose. Proteins in the eluate were detected with goat anti-human IgG (Fc specific), goat anti-human IgG (Cκ

specific), and rabbit anti-actin antibody, followed by HRP-conjugated rabbit anti-goat IgG and HRP-conjugated goat anti-rabbit IgG, respectively. Input and elution samples were resolved by reducing SDS-PAGE. Input represents 10% of the total amount of lysate used for IP.

E. Effect of structural integrity of the constant region on H:L association and formation of the correct antigen-binding site in cytosolic IgGs

If so, is the heavy and light chain linkage of the cytosolic IgG determined entirely by the variable region? To analyze the effect of constant region on H:L association, disulfide bonds were focused , which play an important role in heavy and light chain interaction. The mutant ΔLd-IgG *#H*#L, which cannot form all disulfide bond, was expressed by replacing all cysteine residues in the constant region with serine residues which have same structure with cysteine but one oxygen atom instead of sulfur (Figure 10A). Expression of mutant and wildtype cytosolic IgGs was confirmed by western blotting (Figure 10B) and the assembly of heavy and light chains and antigen-binding activity were confirmed by ELISA (Figure 10C, D). As a result, interaction of heavy chain-light chain and antigen-binding activity were not observed in all mutants. This shows that the constant region of cytosolic IgG may affects the H:L association and antigen-binding activity.

Figure 10. Effect of the absence of disulfide bonds in constant regions on H:L association and correct folding of antigen-binding site in cytosolic IgGs. (A) Schematic representation of Ld-IgG*^#H*^#L. Sharp (#) means disruption of intra-chain disulfide bonds and asterisk (*) means disruption of inter-chain disulfide bonds. (B) Heavy and light chains in lysates of HEK293T cells transiently transfected with KV10 vectors encoding wt ΔLd-IgG and mutant ΔLd-IgG*^#H*^#L were detected by immunoblotting. Heavy chain was detected with goat anti-human IgG (Fc specific) and light chain was detected by goat anti-anti-human IgG (Cκ specific),

followed by HRP-conjugated rabbit anti-goat IgG. (C) ELISA to evaluate association of heavy and light chains was performed using anti-human C as a capture antibody. Lysates of HEK293T cells expressing wt ΔLd-IgG and mutant ΔLd-IgG*^#H*^#L were treated and bound IgGs were detected with AP-conjugated anti-human IgG/Fc antibody. (D) ELISA to evaluate antigen-binding of wt ΔLd-IgG and mutant ΔLd-IgG*^#H*^#L was performed. Lysates of HEK293T cells expressing wt ΔLd-IgG and mutant ΔLd-IgG*^#H*^#L were treated to wells coated with their specific antigens and bound IgGs were detected with AP-conjugated anti-human IgG/Fc antibody.

F. Importance of intra-chain disulfide bonds in H:L association and antigen-binding site formation of cytosolic IgGs

In order to understand disulfide bonds present in the constant region, antibodies were expressed without the variable region and designated as Ig-Cw (Immunoglobulin whole constant region). I constructed a *#CH*#CL mutant that does not form all disulfide bonds and *CH*CL mutant that does not form inter-chain disulfide bonds (Figure 11A). Protein expression was confirmed by immunoblotting (Figure 11B), and expression levels were extremely low when all disulfide bonds were eliminated regardless of the leader sequence. It is presumed that the absence of disulfide bonds has some influence on the stability of the protein. ELISA showed that H:L association was observed in CHCL and *CH*CL, which have all disulfide bonds and intra-chain disulfide bonds, respectively. However, heavy and light chains of *#CH*# CL, which eliminated all disulfide bonds, was not assembled (Figure 11C). These results indicated that intra-chain disulfide bonds are important to association of heavy and light chain.

Figure 11. Effect of intrachain disulfide bonds on H:L association and antigen-binding site formation of cytosolic IgGs. (A) Schematic representation of Ld-Ig-Cw (whole constant region) mutants. Sharp (#) means disruption of intra-chain disulfide bonds and asterisk (*) means disruption of inter-chain disulfide bonds. The black square at the N-terminus of Cw means leader sequence. (B) Heavy and light chains in lysates of HEK293T cells transiently transfected with KV10 vectors encoding Ig-Cw with or without Ld were detected by immunoblotting. Heavy chain was detected with goat anti-human IgG (Fc specific) and light chain was detected by goat anti-human IgG (C specific), followed by HRP-conjugated rabbit anti-goat IgG. (C) ELISA to evaluate association of heavy and light chains was performed using anti-human C as a capture antibody. Lysates of HEK293T cells expressing Ld-Ig-Cw and ΔLd-Ig-Cw constructs were treated and bound Ig-Cw were detected with AP-conjugated anti-human IgG/Fc antibody. Data are presented as mean ± SEM, n = 3.

G. Partial formation of intra-chain disulfide bonds in heavy and light chains of cytosolic IgGs

Cysteine residues in the state of reduced cytosol environment can form disulfide bonds without enzymatic reaction when cells are destroyed and exposed to an oxidizing environment in the air. To exclude this possibility, I compared the differences between the cells after exposure to air for 0 and 12 h by western blotting. The HA or Flag tag were labeled to the carboxy terminal of the heavy or light chain of 3D8 IgG and expressed in the cytosol, and the expression level was confirmed by western blotting (Figure 12A, B). Analysis showed no difference between the 0 and 12 h-exposed samples in the air. When treated with the thiol alkylating agent NEM, some cysteine residues in the IgG were alkylated and the band size was slightly increased (Figure 12B, lane 3,4). The reason for the alkylation of cytosolic IgG by NEM is that some cysteine residues exist in reduced form without forming intra-disulfide bonds. Thus, this suggests that the disulfide bond in the cytosolic IgG is partially formed.

When the antigen-binding capacity of the antibody treated with NEM was determined by ELISA (Figure 12C), in the case of ER-directed IgG, most cysteine residues were already binary and there was little free-thiol. However, cytosolic IgG has a disulfide bond formed only in some cysteine residues, resulting in a decrease in antigen-binding activity due to the presence of alkylated cysteine by NEM. Therefore, it was confirmed that the cysteine residues in the cytosolic IgG partially formed disulfide bonds.

The presence of intra-disulfide bond in cytosolic IgG was confirmed by PEGylation of 3D8 IgG tagged with HA at the carboxy terminus of the heavy chain expressed in the cytosol of HEK293T and treated with NEM to block the free thiol. Then, TCEP was treated to reduce disulfide bonds and free-thiol, newly exposed by TCEP, is detected by PEGylation using

mPEG-MAL followed by detection of IgG heavy chain by western blot. In other words, if there is a disulfide bond in the cytosolic IgG, it can be confirmed by increased molecular weight by western blotting by labeling with PEG (Figure 13A). The NEM present in the cell lysate is removed by a desalting column prior to TCEP treatment because NEM competes with the maleimide group of mPEG-MAL. Western blot results showed that PEGylated IgG heavy chain band, slightly larger than native IgG heavy chain, was observed in lane 1 (Figure 13B). This indicates that disulfide bonds are present in cytosolic IgG.

Figure 12. Absence of spontaneous ex vivo non-enzymatic disulfide bonds formation in cytosolic IgGs. (A) Schematic representation of Ld-3D8 IgG/H-HA and Ld-3D8 IgG/L-Flag proteins. (B) Immunoblotting was performed using lysates of HEK293T cells transiently transfected with KV10 vectors encoding Ld-3D8 IgG/H-HA or Ld-3D8 IgG/L-Flag and prepared in the presence and absence of 100 mM NEM. Proteins were separated by non-reducing SDS-PAGE, both immediately and after 12 h incubation at RT. HA-tagged heavy chain was detected with mouse anti-HA tag antibody and Flag-tagged light chain was detected by moues anti-Flag tag antibody, followed by HRP-conjugated horse anti-mouse IgG. (C) antigen-binding activity of IgGs was evaluated by ELISA. Lysates of HEK293T cells transiently transfected with Ld- or ΔLd-IgGs were prepared in the presence of 100 mM NEM, placed in wells coated with specific antigens. Bound IgGs were detected with AP-conjugated anti-human IgG/Fc. Data are presented as mean ± SEM (n = 6).

Figure 13. Presence of disulfide bonds in the cytosolic IgG. (A) Workflow of the assay.

(B) Immunoblotting. HEK293T cells transfected with a plasmid encoding Ld-3D8 IgG/H-HA were pre-incubated with NEM at a final concentration of 50 mM at 37C for 15 min.

Lysates of cells were then prepared in the presence of 100 mM NEM. NEM was removed by passing the cell lysate through a desalting column to avoid competition with mPEG-MAL.

Lysates were reacted with 5 mM TCEP at 37°C for 1 h and then incubated with 10 mM mPEG-MAL at 37°C for 1 h. PEGylated proteins were separated by reducing SDS-PAGE, and HA labeled heavy chain and PEG were detected by immunoblotting with anti-HA and anti-PEG antibodies, respectively. The PEGylated 3D8 IgG heavy chain is indicated by an arrow.

H. Differences in assembly of IgG1s expressed in the cytosol or via the ER

To determine the difference in disulfide bond formation between cytosolic IgG and ER-directed proteins, in vitro reversible redox reactions were performed. The IgG-expressing HEK293T transfectants were treated with 100 mM of DTT to completely reduce the IgG, and the solution was passed through a desalting column to remove DTT. Following this, micro-dialysis was performed to make the protein fold, and change in the IgG was confirmed by immunoblotting. It is confirmed that DTT could be removed by passing desalting column and dialyzing (Figure 14A). In the DTT-treated sample, all disulfide bonds of IgG were reduced, and a single band was observed regardless of the leader sequence (Figure 14B, lane 3). Upon

문서에서 Structural and Functional Properties of the Recombinant anti-KIFC1 Antibodies Expressed in the Cytosol (페이지 38-0)