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A Monoclonal Antibody Selected for Probing the Folding of Staphylococcal Nuclease and Its N-Terminal Fragments
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
244, 556–560 (1998)
RC978060
A Monoclonal Antibody Selected for Probing the Folding of Staphylococcal Nuclease and Its N-Terminal Fragments Peng Cheng, Jun-Mei Zhou, and Zhen-Quan Guo* National Lab of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, 100101, People’s Republic of China; and *Life Sciences Center, Peking University, Beijing, 100871, People’s Republic of China
Received December 12, 1997
Monoclonal antibody McAb2C9 against Staphylococcal nuclease (SNase R) and its N-terminal fragments was produced and characterized. It was observed that the intact enzyme SNase R and its seven fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) differed in their interactions with McAb2C9. However, the fragments with weak immunoreactivity, such as SNR141 and SNR110, increased ability reacting with McAb2C9 in their partially unfolded state. It suggests that the differences of immunoreactivity among the fragments are due to diverse extent of the exposure of the specific epitope and the conformation of the peptide fragment. The monoclonal antibody McAb2C9 could be a useful probe to investigate the mechanism of folding of SNase R and its N-terminal fragments. q 1998 Academic Press Key Words: Staphylococcal nuclease; monoclonal antibody; protein folding; epitope.
Although the refolding of denatured full-length proteins have provided important information on the mechanism of protein folding, it is not an ideal model, as the refolding of a denatured protein starts from the complete chain, while the folding of the nascent peptide in vivo might begin early during biosynthesis on the ribosome. Studies of a family of peptide fragments with different chain lengths starting from the N-terminal end of a protein seems to provide a better model for protein folding than that study of the refolding of a denatured protein. Therefore, a family of fragments with different chain lengths starting from the N-terminal end of Staphylococcal nuclease R was established as a model to mimic the nascent peptide folding in vitro [1,2]. Abbreviations: ELISA, enzyme-linked immunosorbent assay; GuHCl, guanidine hydrochloride; PBS, phosphate-buffered saline; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SNase R, Staphylococcal nuclease R. 0006-291X/98 $25.00
Staphylococcal Nuclease ( SNase A, EC 3.1.4.7 ) is a small globular protein with 149 amino acid residues containing neither disulfide bonds nor cysteine and has become a good model protein for studies of protein folding [3]. Staphylococcal nuclease R (SNase R) is an analogue of SNase A in which a hexapeptide (DPTVYS) is appended to the N-terminus of SNase A[1]. Besides, seven N-terminal fragments of SNase R named SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52, which extended from residues -6-141, -6135, -6-121, -6-110, -6-102, -6-79 and -6-52 respectively, have been overproduced in E.coli and their conformations in solution have been comparatively studied [1,3]. It is shown that the contents of the secondary structure and enzyme activity of the fragments increase with increasing peptide chain length. However, the significance of the conformation change along the growth of the chain length and the conformation resemblance between the fragments during peptide elongation are not entirely clear. In recent years, many investigations have illustrated the power of monoclonal antibodies as probes to elucidate protein folding both in vitro and in vivo for their ability to recognize and bind their corresponding antigens with high efficacy and specificity [411]. We are also interested in using this method to characterize folding intermediates and to relate the conformation of the intermediates and the N-terminal fragments to that of the native protein. Our experience with Adenylate kinase [12] implied that this would be possible. In present study, a monoclonal antibody McAb2C9 raised with a mixed antigen containing SNase R and its two N-terminal fragments SNR135 and SNR102 was selected and characterized. Experimental results show that the McAb2C9 could be a conformation-dependent monoclonal antibody and a useful tool to probe to what extent Staphylococcal nuclease and its peptide fragments approximate each other in their conformations. MATERIALS AND METHODS Preparation of monoclonal antibody. BALB/c mice of male (8-10 weeks old) were immunized intraperitoneally with a mixed antigen
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containing 20 mg SNase R, 10 mg SNR135 and 10 mg SNR102 emulsified in Freund’s complete adjuvant followed by the booster injection after 3 weeks with similar procedure. Two weeks after the booster immunization, a mixed antigen containing 3 mg SNase R, 1 mg SNR135 and 1 mg SNR102 in 0.1 ml phosphate-buffered saline (PBS) was injected directly into the spleens of mice. Three days later, spleen was removed and spleen cells were fused with mouse myeloma cells SP2/O by PEG (M.W. 1,500). Culture supernatants were screened for specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). Highly positive hybrids were then expanded and subcloned by limiting dilution. The cloned hybridomas were cultured and injected intraperitoneally into mineral oil-primed BALB/c mice. Immuoglobulin fraction in ascitic fluid was first precipitated by caprylic acid and ammonium sulfate successively, then re-dissolved, dialyzed and further purified through anion exchange (DEAE-Sephacel, Pharmacia)[13] and gel-filtration (Sephadex G-150, Pharmacia) chromatography. The antibody was stored in (NH4)2SO4 of 50% saturation. Aliquots of antibodies were centrifuged at 12,000 rpm, and precipitates were then re-solubilized and desalted before use. Competitive ELISA assay. The antibody concentration which gave the sharpest transition on the titer titration curve was used to determine a reasonable coating concentration of antigen SNase R on criteria that the amount of antibody bound was proportional to the amount of total antibody. McAb2C9 and an excess of antigens (SNase R, SNR141, SNR135, SNR121, SNR110, SNR102, SNR79, SNR52) were first incubated in solution at room temperature for 15 min to allow binding to take place. The free antibody remaining in solution was then monitored by a classical ELISA test in a plate coated with SNase R of appropriate amount as determined [14]. Every antigen with molar concentrations 10-fold, 100-fold and 1000-fold higher than that of the antibody was mixed with the antibody respectively. Each assay was done in duplicate. A control containing the antibody alone was prepared at the same time. If the antigen can associate with the antibody easily, the absorbance measured will be much lower than that of the control, and vice versa. Unfolding and association of SNR141 and SNR110 with McAb2C9. SNR141 and SNR110 with 10, 100 and 1000-fold molar concentration higher than the final concentration of McAb2C9 were first incubated with desired concentrations of GuHCl at 47C overnight in 10 mM PBS, pH 7.4, to allow the reaction to reach completion [4]. Then McAb2C9 in PBS with addition of corresponding amount of GuHCl as in unfolding step was mixed with the denatured antigens. After 15 min incubation at room temperature, identical procedure of competitive ELISA test as described in 2.2. was followed. The final concentration of the antibody was 5.0110010 M. Controls for each sample contain 5.0110010 M McAb2C9 and corresponding amount of GuHCl as in unfolding for that sample. Refolding and association of SNR141 and SNR110 with McAb2C9. Fragments SNR141 and SNR110 with 10, 100 and 1000-fold molar concentration higher than the final concentration of McAb2C9 were first incubated with desired concentrations of GuHCl respectively at 47C overnight in 10 mM PBS, pH 7.4, to allow the reaction to reach completion [4]. The refolding was initiated by diluting the denatured protein with PBS containing McAb2C9 to make GuHCl jump to 0.05 M. The final concentration of the antibody was 5.0110010 M. After 15 min incubation at room temperature, a competitive ELISA test was performed. Solutions of 5.0110010 M McAb2C9 in the presence of 0.05 M GuHCl were prepared as controls.
RESULTS AND DISCUSSION
FIG. 1. A450nm versus antibody concentration. The coating amount of antigen SNase R for each well is 0.2 mg. The concentration of McAb2C9 corresponding to ’1’ on the abscissa is 5.0110010 mol/L.
to the method described in 2.2, 0.2 mg/well was determined to be an appropriate coating amount for antigen, when the antibody concentration is 5.0 1 10010 M. Fig. 1 shows that the absorbance of 450 nm (A450nm) in ELISA is proportional to the concentration of antibody. Therefore, the decreased absorbance due to competitive binding of antigens with antibody in solution is proportional to the amount of antibody bound by these antigens. Thus, as shown in equation (1), the ratio, namely ‘binding ratio’, between the decreased value of A450nm (DA) and that of the control (A0) in which the competitor antigens are absent can represent the ratio of the antibody bound by competitive antigen in solution (D[Ab]) and total antibody ([Ab]0).
DA A00A D[Ab] Å Å A0 A0 [Ab]0
[1]
If the competitive antigen with the concentrations 100fold or less than that of the antibody concentrations can lead to 50% decrease of the absorbance at 450nm, the competition is considered to be positive. As shown in Fig.2 and Table 1, SNR141, SNR135 and SNR110 have little effect on the antibody binding to SNase R coated on ELISA plates with increasing amounts. In contrast, SNase R, SNR121, SNR110, SNR79 and SNR52 can bind more than 50% antibodies when their molar concentrations were 100-fold or less than that of the antibody.
Interactions of SNase R and Its Fragments with McAb2C9 in Solution
Interactions of Immobilized SNase R and Its Fragments with McAb2C9
The ability of each peptide fragment binding to McAb2C9 was tested by competitive ELISA. According
Table 2 shows how well the immobilized antigens associate with McAb2C9. The results are similar with
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2
The Binding of Immobilized SNase R and Its Fragments with McAb2C9
OD450nm
FIG. 2. Association of McAb2C9 with SNase R and its N-terminal fragments. The abscissa lg{[Ag]/[Ab]0} represents the logarithm of the ratio between the concentration of antigen ([Ag]) in solution and that of the antibody used ([Ab]0). The vertical axis Binding Ratio (BR) denotes the percentage of the antibody bound by antigen (BRÅD[Ab]/ [Ab]0). The concentration of the antibody McAb2C9 ([Ab]0 ) is 5.0110010 mol/L.
that observed in solution: SNase R and SNR121 bind well to the antibody, while SNR141, SNR135 and SNR110 bind much weakly. The fragments (SNR102, SNR79, SNR52) are too small to be absorbed onto ELISA wells, so this assay was not conducted. The results mentioned above show that the ability of these fragments binding to the antibody do not always increase with increasing length of peptide chains. The differences in immunoreactivity among these antigens revealed their different conformations. Since the antibody associates readily with full-length SNase R as well as the shortest peptide SNR52, it can be deduced that the epitope is located in the sequence of -6-52. So the sequence of the epitope should exist in peptides SNR141, SNR135 and SNR110 as well, their weak reaction with McAb2C9 should be caused by reduced accessibility of the epitope to the antibody other than that the lacking of the epitope.
SNR149
SNR141
SNR135
SNR121
SNR110
0.456
0.024
0.115
0.509
0.031
SNR110, which reacted with McAb2C9 poorly, were examined their immunoreactivity when partially denatured. As shown in Fig.3, when SNR141 was incubated with different concentrations of GuHCl, the binding ratio increased with increasing concentration of GuHCl and attained the maximum at 0.6 M GuHCl. It shows that the greater the antigen unfolded, the more the epitope exposed, and therefore the stronger the antigen-antibody interacted. However, as the GuHCl concentration further increased, the binding ratio decreased to a lower level than unfolded fragments in 0.6M GuHCl but still higher than non-denatured state. For both SNR141 and SNR110 (data was not provided), cases were the same. It seems that partially extended antigen peptide chains, other than the well unfolded chains, were the optimal states to bind to the antibody McAb2C9. Taking account of these results, we conclude that the association of SNR141 and SNR110 with McAb2C9 is surely related to the accessibility of the epitope and the conformation of the fragments. Did the presence of GuHCl affect the antibody activity in this assay? On this purpose, we tested the changes of antibody activity in GuHCl at the same con-
Interactions of Partially Unfolded SNR141 and SNR110 with McAb2C9 In order to determine whether the epitope accessibility resulted in the differences of the binding ability among these peptides, two fragments SNR141 and
TABLE 1
The Binding of SNase R and Its Fragments with McAb2C9 in Solution Antigen
Competition
Antigen
Competition
SNase R SNR141 SNR135 SNR121
/ 0 0 /
SNR110 SNR102 SNR79 SNR52
0 / / /
Fig. 3. Changes of the interaction between SNR141 and McAb2C9 in GuHCl solutions with different concentrations. The molar concentrations of SNR141 are 10-fold (D), 100-fold (s) and 1000fold (l) higher than that of McAb2C9 (5.0110010 M). 558
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Fig. 4. Effect of GuHCl on the activity of antibody McAb2C9. McAb2C9 was first incubated with varying concentrations of GuHCl for 15 min at room temperature and then measured its activity by ELISA.
ditions as mentioned above. McAb2C9 was first incubated with varying concentrations of GuHCl for 15 min, and its activity was then measured by ELISA. The reaction of antigen and antibody reduced slightly when GuHCl concentration increased as shown in Fig. 4. This decrease is caused mainly by the denaturation of antibody, but the conformational changes of SNase R immobilized on ELISA plate caused by GuHCl might also contribute to this decrease. Anyway, both of these have been treated as a background in the denaturation-association assay. Furthermore, each sample has its own control in which corresponding concentration of GuHCl was also added. So by comparing the absorbance of each sample and that of the control in our experiments, the background was eliminated effectively. Thus the changes of association between the partially unfolded fragments and McAb2C9 are surely resulted from the conformational changes of SNR141 and SNR110.
SNR141. The binding ratio differed with varying denaturing states of the fragment. As SNR141 refolding from denatured state in GuHCl higher than 0.8 M in which its interaction with antibody was poor (Fig. 3), the association increased cooperatively with increasing GuHCl concentration. When GuHCl concentration was up to 1.0 M, SNR141 trapped almost all the antibody in solution during its refolding (Fig.5). It demonstrates clearly that the refolding intermediates of the fragment can bind with antibody strongly. When SNR141 refolded in GuHCl whose concentrations were in the range of 0.05-0.4 M to the final state (GuHCl 0.05 M), there is no discernible change for the antigen-antibody combination. Yet it is obvious that if the fragments were denatured in guanidine between 0.4 M to 0.8 M and then refold, the binding ratio decreased and reduced to the minimum at 0.8 M GuHCl. Comparing the results with denatured state in Fig.4, we suggested that this reduced binding ability might be caused by the aggregation of the partially unfolded peptide in the presence of mild concentrations of GuHCl. It is very likely that the self-associating sites of the peptide chains are related to the antigen-antibody binding sites and thus lead to the decrease of the interaction between the fragment and the antibody molecules. From the curves in Fig.5, it can also be found that, as the binding ratio increased, the effect of the amount of the antigen became faint. When the concentrations of GuHCl were up to 1.0 M or higher, the three curves with different
Changes of Interaction of SNR141 and SNR110 with McAb2C9 during Refolding As described in 3.3, both SNR141 and SNR110 have optimal conformations to associate with McAb2C9 when unfolded in 0.6 M GuHCl. However, how the optimal conformation looks like and whether it is involved in the folding process are not clear yet. To search for answering these questions, McAb2C9 was investigated its reaction with SNR141 and SNR110 during refolding from different denaturing states to the same state in which the final guanidine concentration is 0.05M. As shown in Fig.5, marked increase of antigenantibody reaction was observed during refolding of
FIG. 5. Changes of interaction between SNR141 and McAb2C9 during refolding. SNR141 was first denatured in different concentrations of GuHCl as indicated on the abscissa and then refolded by dilution as described in the text. The molar concentrations of SNR141 are 10-fold (D),100-fold (s) and 1000-fold (l) higher than that of McAb2C9 (5.0110010 M). The final concentration of GuHCl in each sample was 0.05 M. The changes of binding ratio were compared with the control which contained only McAb2C9 (5.0110010 M) and GuHCl (0.05M) but no SNR141.
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concentrations of SNR141 are almost overlapped completely. This suggested that the ability of fragment SNR141 reacting with the antibody increased to such an extent that even with only 10-fold excess amount it can still trap the antibody properly. As for the fragment SNR110, very similar results were obtained (data was not shown). This result indicated that the antibody not just captured the antigen in unfolded state as long as the epitope was exposed but appeared to recognize a folding intermediate involved in the refolding process. Thus McAb2C9 will be a useful tool for investigating the regain of the specific conformation for antigen-antibody binding during renaturation of SNase R. In some cases, monoclonal antibody may hinder or affect some of the folding steps which occur after its binding to the antigen [4]. Indication on this aspect for McAb2C9 and identifying the actual conformation recognized by the antibody will require more detailed structural studies. ACKNOWLEDGMENTS We thank Professor C.-L. Tsou and Prof. G.-Z. Jing for their advice and encouragement. We are grateful to B. Zhou, Z.-H. Huang, K.-G. Tian and J.-K. Song for providing SNase R and the N-terminal peptide fragments. This work was supported in part by the Pandeng Project of the China Commission for Science and technology.
REFERENCES 1. Jing, G. Z., Zhou, B., Xie, L., Liu, L. J., and Liu, Z. G. (1995) Biochim. Biophys. Acta 1250, 189–196. 2. Tsou, C. L. (1988) Biochemistry 27, 1809–1812. 3. Zhou, B., and Jing, G. Z. (1996) J. Biochem. 120, 881–886. 4. Speed, M. A., Morshead, T., Wang, D. I. C., and King, J. (1997) Protein Sci. 6, 99–108. 5. Solomon, B., Koppel, R., Hanan, E., and Katzav, T. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 452–455. 6. Friguet, B., Djavadi-Ohaniance, L., King, J., and Goldberg, M. E. (1994) J. Biol. Chem. 269, 15945–15949. 7. Fedorov, A. N., Friguet, B., Djavadi-Ohaniance, L., Alakhov, Y. B., and Goldberg, M. E. (1992) J. Mol. Biol. 228, 351–358. 8. Blond, S., and Goldberg, M. E. (1991) Biochemistry 29, 2409– 2417. 9. Blond, S., and Goldberg, M. E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1147–1151. 10. Goldberg, M. E. (1991) Trends Biochem. Sci. 16, 358–362. 11. Friguet, B., Djavadi-Ohaniance, L., Haase-Pettingell, C. A., King, J., and Goldberg, M. E. (1990) J. Biol. Chem. 265, 10347– 10351. 12. Wang, X. D., Zhou, J. M., and Guo, Z. Q. Sciences in China (in press). 13. Smith, T. J. (1993) Methods Cell Biol. 37, 75–83. 14. Friguet, B., Djavadi-Ohaniance, L., and Goldberg, M. E. (1984) Mol. Immunol. 21, 673–677.
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244, 556–560 (1998)
RC978060
A Monoclonal Antibody Selected for Probing the Folding of Staphylococcal Nuclease and Its N-Terminal Fragments Peng Cheng, Jun-Mei Zhou, and Zhen-Quan Guo* National Lab of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, 100101, People’s Republic of China; and *Life Sciences Center, Peking University, Beijing, 100871, People’s Republic of China
Received December 12, 1997
Monoclonal antibody McAb2C9 against Staphylococcal nuclease (SNase R) and its N-terminal fragments was produced and characterized. It was observed that the intact enzyme SNase R and its seven fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) differed in their interactions with McAb2C9. However, the fragments with weak immunoreactivity, such as SNR141 and SNR110, increased ability reacting with McAb2C9 in their partially unfolded state. It suggests that the differences of immunoreactivity among the fragments are due to diverse extent of the exposure of the specific epitope and the conformation of the peptide fragment. The monoclonal antibody McAb2C9 could be a useful probe to investigate the mechanism of folding of SNase R and its N-terminal fragments. q 1998 Academic Press Key Words: Staphylococcal nuclease; monoclonal antibody; protein folding; epitope.
Although the refolding of denatured full-length proteins have provided important information on the mechanism of protein folding, it is not an ideal model, as the refolding of a denatured protein starts from the complete chain, while the folding of the nascent peptide in vivo might begin early during biosynthesis on the ribosome. Studies of a family of peptide fragments with different chain lengths starting from the N-terminal end of a protein seems to provide a better model for protein folding than that study of the refolding of a denatured protein. Therefore, a family of fragments with different chain lengths starting from the N-terminal end of Staphylococcal nuclease R was established as a model to mimic the nascent peptide folding in vitro [1,2]. Abbreviations: ELISA, enzyme-linked immunosorbent assay; GuHCl, guanidine hydrochloride; PBS, phosphate-buffered saline; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SNase R, Staphylococcal nuclease R. 0006-291X/98 $25.00
Staphylococcal Nuclease ( SNase A, EC 3.1.4.7 ) is a small globular protein with 149 amino acid residues containing neither disulfide bonds nor cysteine and has become a good model protein for studies of protein folding [3]. Staphylococcal nuclease R (SNase R) is an analogue of SNase A in which a hexapeptide (DPTVYS) is appended to the N-terminus of SNase A[1]. Besides, seven N-terminal fragments of SNase R named SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52, which extended from residues -6-141, -6135, -6-121, -6-110, -6-102, -6-79 and -6-52 respectively, have been overproduced in E.coli and their conformations in solution have been comparatively studied [1,3]. It is shown that the contents of the secondary structure and enzyme activity of the fragments increase with increasing peptide chain length. However, the significance of the conformation change along the growth of the chain length and the conformation resemblance between the fragments during peptide elongation are not entirely clear. In recent years, many investigations have illustrated the power of monoclonal antibodies as probes to elucidate protein folding both in vitro and in vivo for their ability to recognize and bind their corresponding antigens with high efficacy and specificity [411]. We are also interested in using this method to characterize folding intermediates and to relate the conformation of the intermediates and the N-terminal fragments to that of the native protein. Our experience with Adenylate kinase [12] implied that this would be possible. In present study, a monoclonal antibody McAb2C9 raised with a mixed antigen containing SNase R and its two N-terminal fragments SNR135 and SNR102 was selected and characterized. Experimental results show that the McAb2C9 could be a conformation-dependent monoclonal antibody and a useful tool to probe to what extent Staphylococcal nuclease and its peptide fragments approximate each other in their conformations. MATERIALS AND METHODS Preparation of monoclonal antibody. BALB/c mice of male (8-10 weeks old) were immunized intraperitoneally with a mixed antigen
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containing 20 mg SNase R, 10 mg SNR135 and 10 mg SNR102 emulsified in Freund’s complete adjuvant followed by the booster injection after 3 weeks with similar procedure. Two weeks after the booster immunization, a mixed antigen containing 3 mg SNase R, 1 mg SNR135 and 1 mg SNR102 in 0.1 ml phosphate-buffered saline (PBS) was injected directly into the spleens of mice. Three days later, spleen was removed and spleen cells were fused with mouse myeloma cells SP2/O by PEG (M.W. 1,500). Culture supernatants were screened for specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). Highly positive hybrids were then expanded and subcloned by limiting dilution. The cloned hybridomas were cultured and injected intraperitoneally into mineral oil-primed BALB/c mice. Immuoglobulin fraction in ascitic fluid was first precipitated by caprylic acid and ammonium sulfate successively, then re-dissolved, dialyzed and further purified through anion exchange (DEAE-Sephacel, Pharmacia)[13] and gel-filtration (Sephadex G-150, Pharmacia) chromatography. The antibody was stored in (NH4)2SO4 of 50% saturation. Aliquots of antibodies were centrifuged at 12,000 rpm, and precipitates were then re-solubilized and desalted before use. Competitive ELISA assay. The antibody concentration which gave the sharpest transition on the titer titration curve was used to determine a reasonable coating concentration of antigen SNase R on criteria that the amount of antibody bound was proportional to the amount of total antibody. McAb2C9 and an excess of antigens (SNase R, SNR141, SNR135, SNR121, SNR110, SNR102, SNR79, SNR52) were first incubated in solution at room temperature for 15 min to allow binding to take place. The free antibody remaining in solution was then monitored by a classical ELISA test in a plate coated with SNase R of appropriate amount as determined [14]. Every antigen with molar concentrations 10-fold, 100-fold and 1000-fold higher than that of the antibody was mixed with the antibody respectively. Each assay was done in duplicate. A control containing the antibody alone was prepared at the same time. If the antigen can associate with the antibody easily, the absorbance measured will be much lower than that of the control, and vice versa. Unfolding and association of SNR141 and SNR110 with McAb2C9. SNR141 and SNR110 with 10, 100 and 1000-fold molar concentration higher than the final concentration of McAb2C9 were first incubated with desired concentrations of GuHCl at 47C overnight in 10 mM PBS, pH 7.4, to allow the reaction to reach completion [4]. Then McAb2C9 in PBS with addition of corresponding amount of GuHCl as in unfolding step was mixed with the denatured antigens. After 15 min incubation at room temperature, identical procedure of competitive ELISA test as described in 2.2. was followed. The final concentration of the antibody was 5.0110010 M. Controls for each sample contain 5.0110010 M McAb2C9 and corresponding amount of GuHCl as in unfolding for that sample. Refolding and association of SNR141 and SNR110 with McAb2C9. Fragments SNR141 and SNR110 with 10, 100 and 1000-fold molar concentration higher than the final concentration of McAb2C9 were first incubated with desired concentrations of GuHCl respectively at 47C overnight in 10 mM PBS, pH 7.4, to allow the reaction to reach completion [4]. The refolding was initiated by diluting the denatured protein with PBS containing McAb2C9 to make GuHCl jump to 0.05 M. The final concentration of the antibody was 5.0110010 M. After 15 min incubation at room temperature, a competitive ELISA test was performed. Solutions of 5.0110010 M McAb2C9 in the presence of 0.05 M GuHCl were prepared as controls.
RESULTS AND DISCUSSION
FIG. 1. A450nm versus antibody concentration. The coating amount of antigen SNase R for each well is 0.2 mg. The concentration of McAb2C9 corresponding to ’1’ on the abscissa is 5.0110010 mol/L.
to the method described in 2.2, 0.2 mg/well was determined to be an appropriate coating amount for antigen, when the antibody concentration is 5.0 1 10010 M. Fig. 1 shows that the absorbance of 450 nm (A450nm) in ELISA is proportional to the concentration of antibody. Therefore, the decreased absorbance due to competitive binding of antigens with antibody in solution is proportional to the amount of antibody bound by these antigens. Thus, as shown in equation (1), the ratio, namely ‘binding ratio’, between the decreased value of A450nm (DA) and that of the control (A0) in which the competitor antigens are absent can represent the ratio of the antibody bound by competitive antigen in solution (D[Ab]) and total antibody ([Ab]0).
DA A00A D[Ab] Å Å A0 A0 [Ab]0
[1]
If the competitive antigen with the concentrations 100fold or less than that of the antibody concentrations can lead to 50% decrease of the absorbance at 450nm, the competition is considered to be positive. As shown in Fig.2 and Table 1, SNR141, SNR135 and SNR110 have little effect on the antibody binding to SNase R coated on ELISA plates with increasing amounts. In contrast, SNase R, SNR121, SNR110, SNR79 and SNR52 can bind more than 50% antibodies when their molar concentrations were 100-fold or less than that of the antibody.
Interactions of SNase R and Its Fragments with McAb2C9 in Solution
Interactions of Immobilized SNase R and Its Fragments with McAb2C9
The ability of each peptide fragment binding to McAb2C9 was tested by competitive ELISA. According
Table 2 shows how well the immobilized antigens associate with McAb2C9. The results are similar with
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The Binding of Immobilized SNase R and Its Fragments with McAb2C9
OD450nm
FIG. 2. Association of McAb2C9 with SNase R and its N-terminal fragments. The abscissa lg{[Ag]/[Ab]0} represents the logarithm of the ratio between the concentration of antigen ([Ag]) in solution and that of the antibody used ([Ab]0). The vertical axis Binding Ratio (BR) denotes the percentage of the antibody bound by antigen (BRÅD[Ab]/ [Ab]0). The concentration of the antibody McAb2C9 ([Ab]0 ) is 5.0110010 mol/L.
that observed in solution: SNase R and SNR121 bind well to the antibody, while SNR141, SNR135 and SNR110 bind much weakly. The fragments (SNR102, SNR79, SNR52) are too small to be absorbed onto ELISA wells, so this assay was not conducted. The results mentioned above show that the ability of these fragments binding to the antibody do not always increase with increasing length of peptide chains. The differences in immunoreactivity among these antigens revealed their different conformations. Since the antibody associates readily with full-length SNase R as well as the shortest peptide SNR52, it can be deduced that the epitope is located in the sequence of -6-52. So the sequence of the epitope should exist in peptides SNR141, SNR135 and SNR110 as well, their weak reaction with McAb2C9 should be caused by reduced accessibility of the epitope to the antibody other than that the lacking of the epitope.
SNR149
SNR141
SNR135
SNR121
SNR110
0.456
0.024
0.115
0.509
0.031
SNR110, which reacted with McAb2C9 poorly, were examined their immunoreactivity when partially denatured. As shown in Fig.3, when SNR141 was incubated with different concentrations of GuHCl, the binding ratio increased with increasing concentration of GuHCl and attained the maximum at 0.6 M GuHCl. It shows that the greater the antigen unfolded, the more the epitope exposed, and therefore the stronger the antigen-antibody interacted. However, as the GuHCl concentration further increased, the binding ratio decreased to a lower level than unfolded fragments in 0.6M GuHCl but still higher than non-denatured state. For both SNR141 and SNR110 (data was not provided), cases were the same. It seems that partially extended antigen peptide chains, other than the well unfolded chains, were the optimal states to bind to the antibody McAb2C9. Taking account of these results, we conclude that the association of SNR141 and SNR110 with McAb2C9 is surely related to the accessibility of the epitope and the conformation of the fragments. Did the presence of GuHCl affect the antibody activity in this assay? On this purpose, we tested the changes of antibody activity in GuHCl at the same con-
Interactions of Partially Unfolded SNR141 and SNR110 with McAb2C9 In order to determine whether the epitope accessibility resulted in the differences of the binding ability among these peptides, two fragments SNR141 and
TABLE 1
The Binding of SNase R and Its Fragments with McAb2C9 in Solution Antigen
Competition
Antigen
Competition
SNase R SNR141 SNR135 SNR121
/ 0 0 /
SNR110 SNR102 SNR79 SNR52
0 / / /
Fig. 3. Changes of the interaction between SNR141 and McAb2C9 in GuHCl solutions with different concentrations. The molar concentrations of SNR141 are 10-fold (D), 100-fold (s) and 1000fold (l) higher than that of McAb2C9 (5.0110010 M). 558
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Fig. 4. Effect of GuHCl on the activity of antibody McAb2C9. McAb2C9 was first incubated with varying concentrations of GuHCl for 15 min at room temperature and then measured its activity by ELISA.
ditions as mentioned above. McAb2C9 was first incubated with varying concentrations of GuHCl for 15 min, and its activity was then measured by ELISA. The reaction of antigen and antibody reduced slightly when GuHCl concentration increased as shown in Fig. 4. This decrease is caused mainly by the denaturation of antibody, but the conformational changes of SNase R immobilized on ELISA plate caused by GuHCl might also contribute to this decrease. Anyway, both of these have been treated as a background in the denaturation-association assay. Furthermore, each sample has its own control in which corresponding concentration of GuHCl was also added. So by comparing the absorbance of each sample and that of the control in our experiments, the background was eliminated effectively. Thus the changes of association between the partially unfolded fragments and McAb2C9 are surely resulted from the conformational changes of SNR141 and SNR110.
SNR141. The binding ratio differed with varying denaturing states of the fragment. As SNR141 refolding from denatured state in GuHCl higher than 0.8 M in which its interaction with antibody was poor (Fig. 3), the association increased cooperatively with increasing GuHCl concentration. When GuHCl concentration was up to 1.0 M, SNR141 trapped almost all the antibody in solution during its refolding (Fig.5). It demonstrates clearly that the refolding intermediates of the fragment can bind with antibody strongly. When SNR141 refolded in GuHCl whose concentrations were in the range of 0.05-0.4 M to the final state (GuHCl 0.05 M), there is no discernible change for the antigen-antibody combination. Yet it is obvious that if the fragments were denatured in guanidine between 0.4 M to 0.8 M and then refold, the binding ratio decreased and reduced to the minimum at 0.8 M GuHCl. Comparing the results with denatured state in Fig.4, we suggested that this reduced binding ability might be caused by the aggregation of the partially unfolded peptide in the presence of mild concentrations of GuHCl. It is very likely that the self-associating sites of the peptide chains are related to the antigen-antibody binding sites and thus lead to the decrease of the interaction between the fragment and the antibody molecules. From the curves in Fig.5, it can also be found that, as the binding ratio increased, the effect of the amount of the antigen became faint. When the concentrations of GuHCl were up to 1.0 M or higher, the three curves with different
Changes of Interaction of SNR141 and SNR110 with McAb2C9 during Refolding As described in 3.3, both SNR141 and SNR110 have optimal conformations to associate with McAb2C9 when unfolded in 0.6 M GuHCl. However, how the optimal conformation looks like and whether it is involved in the folding process are not clear yet. To search for answering these questions, McAb2C9 was investigated its reaction with SNR141 and SNR110 during refolding from different denaturing states to the same state in which the final guanidine concentration is 0.05M. As shown in Fig.5, marked increase of antigenantibody reaction was observed during refolding of
FIG. 5. Changes of interaction between SNR141 and McAb2C9 during refolding. SNR141 was first denatured in different concentrations of GuHCl as indicated on the abscissa and then refolded by dilution as described in the text. The molar concentrations of SNR141 are 10-fold (D),100-fold (s) and 1000-fold (l) higher than that of McAb2C9 (5.0110010 M). The final concentration of GuHCl in each sample was 0.05 M. The changes of binding ratio were compared with the control which contained only McAb2C9 (5.0110010 M) and GuHCl (0.05M) but no SNR141.
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concentrations of SNR141 are almost overlapped completely. This suggested that the ability of fragment SNR141 reacting with the antibody increased to such an extent that even with only 10-fold excess amount it can still trap the antibody properly. As for the fragment SNR110, very similar results were obtained (data was not shown). This result indicated that the antibody not just captured the antigen in unfolded state as long as the epitope was exposed but appeared to recognize a folding intermediate involved in the refolding process. Thus McAb2C9 will be a useful tool for investigating the regain of the specific conformation for antigen-antibody binding during renaturation of SNase R. In some cases, monoclonal antibody may hinder or affect some of the folding steps which occur after its binding to the antigen [4]. Indication on this aspect for McAb2C9 and identifying the actual conformation recognized by the antibody will require more detailed structural studies. ACKNOWLEDGMENTS We thank Professor C.-L. Tsou and Prof. G.-Z. Jing for their advice and encouragement. We are grateful to B. Zhou, Z.-H. Huang, K.-G. Tian and J.-K. Song for providing SNase R and the N-terminal peptide fragments. This work was supported in part by the Pandeng Project of the China Commission for Science and technology.
REFERENCES 1. Jing, G. Z., Zhou, B., Xie, L., Liu, L. J., and Liu, Z. G. (1995) Biochim. Biophys. Acta 1250, 189–196. 2. Tsou, C. L. (1988) Biochemistry 27, 1809–1812. 3. Zhou, B., and Jing, G. Z. (1996) J. Biochem. 120, 881–886. 4. Speed, M. A., Morshead, T., Wang, D. I. C., and King, J. (1997) Protein Sci. 6, 99–108. 5. Solomon, B., Koppel, R., Hanan, E., and Katzav, T. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 452–455. 6. Friguet, B., Djavadi-Ohaniance, L., King, J., and Goldberg, M. E. (1994) J. Biol. Chem. 269, 15945–15949. 7. Fedorov, A. N., Friguet, B., Djavadi-Ohaniance, L., Alakhov, Y. B., and Goldberg, M. E. (1992) J. Mol. Biol. 228, 351–358. 8. Blond, S., and Goldberg, M. E. (1991) Biochemistry 29, 2409– 2417. 9. Blond, S., and Goldberg, M. E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1147–1151. 10. Goldberg, M. E. (1991) Trends Biochem. Sci. 16, 358–362. 11. Friguet, B., Djavadi-Ohaniance, L., Haase-Pettingell, C. A., King, J., and Goldberg, M. E. (1990) J. Biol. Chem. 265, 10347– 10351. 12. Wang, X. D., Zhou, J. M., and Guo, Z. Q. Sciences in China (in press). 13. Smith, T. J. (1993) Methods Cell Biol. 37, 75–83. 14. Friguet, B., Djavadi-Ohaniance, L., and Goldberg, M. E. (1984) Mol. Immunol. 21, 673–677.
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