Russian Journal of Bioorganic Chemistry, Vol. 31, No. 5, 2005, pp. 433–438. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 5, 2005, pp. 482–487. Original Russian Text Copyright © 2005 by Sukhacheva, Tul’kina, Karger, Sheveleva, Stratonova, Dorokhov.
Mapping of Epitopes of the Recombinant Movement Protein of the Tobacco Mosaic Virus with the Use of Monoclonal Antibodies E. A. Sukhacheva*, L. G. Tul’kina**, E. M. Karger**, A. A. Sheveleva**, N. V. Stratonova*, and Yu. L. Dorokhov**, 1 * Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117871 Russia ** Virology Department and Belozerskii Institute of Physicochemical Biology, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia Received November 26, 2004; in final form, April 5, 2005
Abstract—The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. MP fulfils its function while interacting with host cell factors on the whole way of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfilling the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MPr) were obtained. MAs were tested by the immunoblotting and ELISA with the use of deletion variations of MPr. The epitopes of TMV MPr that recognize specific MAs were determined. Key words: movement protein, tobacco mosaic virus, monoclonal antibodies 1
INTRODUCTION
Plasmodesmata of plants form a cytoplasmic network that provides an intercellular transport of macromolecules.2 Plant viruses and, particularly TMV as the most studied representative of the plant RNA-containing viruses, can be used as models in studying both the role of cellular and viral genome in the intracellular transport of the viral RNA in plant and the general mechanisms of macromolecular transport. A growing number of facts indicate that the cellular proteins involved in the normal cellular metabolism also participate in the replication and transport of the viral genome. 1 Corresponding
author; phone: +7 (095) 939-3360; e-mail:
[email protected] 2 Abbreviations: ER, endoplasmic reticulum; GFP , recombinant r green fluorescent protein; IPTG, isopropyl β-D-thiogalactopyranoside; HAT, the solution containing 10–4 M hypoxanthine, 10–7 M aminopterin, and 1.6 × 10–5 M thymidine; HT, the solution containing 10–4 M hypoxanthine and 1.6 × 10–5 M thymidine; MA, monoclonal antibody; MP, movement protein; MPr and MPr∆, recombinant movement protein and its deletion mutants; PME, pectin methylesterase (tobacco); MP-(1-30)-GFPr, recombinant hybrid protein consisting of the first 30 N-terminal amino acid residues of TMV-MP connected with GFPr through a hexapeptide bridge; PAT, murine polyclonal antibodies to TMV-MP; PVDF, polyvinylidene difluoride membrane; and TMV, tobacco mosaic virus.
The TMV MP is responsible for the intercellular transport of the viral RNA [1]. TMV MP is a phosphoprotein that consists of 268 aa and is distinguished by a high content of Lys and Arg residues. MP fulfils its functions in association with cellular factors. It is accumulated in plasmodesmata of plants infected by TMV [2], is associated with ER membranes [3, 4], and is colocalized with the microtubules of a cytoskeleton [5]. The MP ability to increase the plasmodesmata permeability (SEL) [6, 7] and its availability for phosphorylation by cellular protein kinases [8] are important for the understanding of its function. TMV MP can specifically bind the viral RNA, and this fact gave rise to the widely approved hypothesis that the RNA–MP complex is a transport form of TMV [9–11]. The conformation of TMV MP functioning inside cell is still not studied. We describe here the preparation of a panel of murine monoclonal antibodies to the recombinant TMV MPr and the mapping of the MPr epitopes in order to find surface determinants of TMV MP. RESULTS AND DISCUSSION The recombinant protein, which, unlike the natural protein, is not phosphorylated and contains the (His)6 sequence on its N-terminus, was used as an immunogen. It is important to note that MPr behaves as a hydro-
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268
åê
åê 30
NT30
268 268
98
19K
268
134 16K 1
54 70
268
28K-1 1
268
75 90
28K-2 1
96 109
268
1
110 126
268
28K-3 Del3 129
1
186
268
Del4 Del10 CT46 CT11
185
1
åê∆1-97 åê∆1-133 åê∆55-69 åê∆76-89 åê∆97-108 åê∆111-125 åê∆130-185 åê∆184-268
222
1
åê∆1-29
åê∆223-268 257
1
åê∆258-268
Fig. 1. Scheme of the deletion mutants of TMV MPr used for the mapping of the MP epitopes by monoclonal antibodies.
phobic and easily aggregated proteins in aqueous solutions [12]. Therefore, TMV MP was dissolved in 2 M urea for the immunization and subsequent production of MAs to its linear determinants. Fusion of the myeloma cells of the sp2/0 line with the splenocytes of an immune mouse gave eight stable hybridoma lines that produced MAs to the full-size MPr. All the MAs were specifically bound to the homol-
ogous antigen in ELISA and immunoblotting. The variants of MPr with deletions in the N-terminal, C-terminal, and internal regions were used for the more exact localization of the binding sites of the produced MAs (Fig. 1). The results of mapping of the MPr epitopes by these MAs are given in Table 1 and Fig. 2. As follows from these data, we failed to obtain the MAs specific to the N-terminus of MPr (1–30 aa) at the
Table 1. Epitope mapping of TMV M P *r Deletion mutants of TMV MP
MA to MPr
Isotype
MP
1E1 1D8 1A10 3B10 4D9 7C7 7D9 8D12
IgG1 IgG1 IgG2a IgG1 IgG1 IgG1 IgG1 IgG1
+ + + + + + + +
CT11 CT46 Del10 Del4 28K-1 28K-2 28K-3 Del3 Nt30 + + + + + + – –
+ + – + + + – –
– + – + – + – –
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + – + – + +
+ + + – + – + +
19K
16K
PME
Epitope MP
+ – + + + + + +
+ – + – + – + +
– – – – – – – –
186–222 76–89 223–257 98–129 186–222 98–129 258–268 258–268
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+ + + + + + + +
* The signs + and – denote the positive and negative reactions at immunoblotting; PME is a negative control. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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(‡)
PME
Del10
435
Del4
Del3
19K
16K
NT30
CT46
CT11
MPr
PME
Del10
Del4
Del3
19K
16K
NT30
CT46
CT11
MPr
MAPPING OF EPITOPES OF THE RECOMBINANT MOVEMENT PROTEIN
(b)
Fig. 2. Immunoblotting (typical pattern) on the basis of which positive (+) or negative (–) binding of MA to the deletion variant of TMV MPr was detected. The results for (a) MA 7D9 (epitop 258–268 aa) and (b) MA 1D8 (epitop 76–89 aa) are given.
first stage of our study. Therefore, we used the recombinant hybrid protein containing 30 N-terminal amino acid residues of the TMV MP connected to the GFP amino acid sequence through a GGEFPR peptide bridge and the (His)6 sequence at the ë-terminus of the hybrid molecule as an immunogen for the preparation of MA to the N-terminus of MP at the next stage of our study. We obtained 15 hybridoma lines producing MAs that specifically interacted with MP-(1–30)-GFPr and did not recognize GFPr (Table 2). Only four of the 15 MAs proved to be specific to the MP amino acid sequence. A further analysis of MAs to MP-(1–30)-
GFPr was carried out using the additionally prepared recombinant proteins: MP with six His residues on its C-terminus and mutant MP-(1-30)-GTP with the deletion of the second amino acid residue. We found that the MAs that do not interact with the MP amino acid sequence were produced to the hexapeptide bridge connecting the MP-(1-30) fragment with GTP (data not shown). Thus, we prepared MAs to the N-terminal, C-terminal, and internal regions of TMV MPr (Fig. 3). Further, we plan to map the surface of the intracellular MP and to study its post-translational modifications.
Table 2. Selection of MAs specific to the N-terminal fragment of TMV MP* Methods of MA testing MA to MP-(1–30)-GFPr 1E4 1E5 1B6 1F3 2E9 2H11 3B11 3D1 3G1 3E1 3F12 4A3 4F4 5B12 5D8
MA isotype IgG1 IgG2b IgG2b N.d. N.d. N.d. N.d. N.d. N.d. N.d. N.d. IgG1 N.d. IgG2b N.d.
EIA
immunoblotting
MP-(1–30)-GFPr
TMV-MPr
+ + + + + + + + + + + + + + +
+ – + – – – – – – – – + – – –
MP-(1–30)-GFPr TMV-MPr + + + + + + + + + + + + + + +
+ – + + – – – – – – – + – – –
* The signs + and – denote the positive and negative reactions at immunoblotting; N.d., not determined. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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19K
GFPr
– – – – – – – – – – – – – – –
– – – – – – – – – – – – – – –
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30
4A3 1B6
78
89 98
1D8
129
3B10 7C7
186
222 223 257
1E1 4D9
268
1A10 8D12 7D9
Fig. 3. Correspondence of the obtained MA to the MPr epitopes.
EXPERIMENTAL The DMEM, HAT-DMEM, and HT-DMEM media, calf embryo serum, glutamine, and Freund’s adjuvant were from Life Technologies (United States); rabbit antimouse antibodies conjugated with alkaline phosphatase, pristane, Tween-20, IPTG, and polyacrylamide, from Sigma (United States); polyethylene glycol 1500 and dimethylsulfoxide, from Merck (Germany); bovine serum albumin, from Serva (Germany); protein-G-Sepharose, from Pharmacia (Sweden); nickel-chelate sorbent and plastic columns for purification of recombinant proteins, from Qiagen (Germany); PVDF-membrane and ECL system for blot coloring, from Amersham; 96-well plates for immunoassay, 24well and 96-well plates and flasks for cell cultures, from Costar (Netherlands); and rabbit antibodies specific to various subclasses of the murine MAs, from Calbiochem). Deoxyribooligonucleotide primers were synthesized by Syntol Company (Russia) and GibcoBRL Company (Germany). The following enzymes were used in this study: restriction endonucleases (Sibenzim, Novosibirsk and Fermentas, Vilnius), DNA ligase of T4 phage (Fermentas), and Taq-polymerase (Sibenzim). The genes of the TMV MPr deletion mutants were cloned into the plasmid vectors (Qiagen, Germany): pQE-30 at the BamHI and PstI restriction sites, pQE-60 at the NcoI and BamHI restriction sites, and pQE-70 at the SphI and BamHI restriction sites. The hexahistidine sequence was placed on the N-terminus of the recombinant protein upon cloning of the protein gene into pQE60 and on the C-terminus upon its cloning into pQE-70. We described the preparation of MPr previously [13]. The genes of C-terminal deletion mutants of MPr were prepared by PCR on the pQE-MP template [12] using the common direct primer P1 (Table 3) and the following reverse primers: P3 for CT11, P4 for CT46, and P5 for Del10. The genes of the N-terminal deletion mutants of MPr were also prepared by PCR on the pQEMP template using the common reverse primer P2 and the following direct primers: P6 for NT30, P7 for 19K, and P8 for 16K. The genes of the TMV MPr mutants with the internal deletions were prepared by the method of overlapping PCR. The PCR was carried out on the pQE-MP template with the following two pairs of primers: P1 and P9 and P10 and P2 in the case of Del3; P1 and P11 and P12 and P2 in the case of 28K-1; P1 and P13 and P14 and P2 in the case of 28K-2; and P1 and P15 and P16 and P2 in the case of 28K-3. New PCR
with P1 and P2 primers were performed on the basis of two PCR products, and the products obtained from the last PCR were cloned into the pQE-30 at the BamHI and PstI unique sites. The gene of the recombinant GFP was prepared by PCR on the pFF-IRES-MP-GFP template (the plasmid was a kind gift of E.V. Skuratov from the Moscow State University) from the P17 direct primer and the P18 reverse primer and cloned into the pQE-30 at the BamHI and PstI restriction sites [(His)6-GFPr]. Two parts of the gene of the MP-(1–30)-GFPr hybrid protein were prepared by PCR on the pFF-IRES-MP-GFP template from the corresponding pairs of primers: P19 and P20 for MP-(1–30) and P20 and P21 for GFP. The sequence of hexapeptide bridge was encoded in the nucleotide sequences of the P21 and P22 primers (Table 3, marked with boldface). The reaction products were ligated with the reduction of the EcoRI site and cloned into the pQE-70 at the SphI and BamHI restriction sites. The gene of the MP-(1–30)-GFPr hybrid protein (it differs from MP-(1–30)-GFP by the deletion of the second amino acid residue) was prepared by PCR on the MP-(1–30)-GFPr template using the P23 and P22 primers and cloned into the pQE-70 in the NcoI and BamHI restriction sites. The gene of the recombinant TMV MP with six His residues on the C-terminus was prepared by PCR on the pFF-IRES-MP-GFP template using the P23 and P24 primers and cloned into pQE-60 at the NcoI and BamHI restriction sites. After the cloning into pQE-30, pQE-60, and pQE-70, all the amplicons were identified by sequencing. The gene of the TMV-MPrDel4 deletion mutant was kindly presented by Dr. V. Citovsky (State University of New York). The E. coli cells of the M15 strain (DIAGEN) were transformed by the recombinant plasmid, and expression of the cloned gene was induced by IPTG. The recombinant proteins containing (His)6 sequence were separated by chromatography on a nickel-chelate sorbent [14] with the subsequent electrophoretic analysis on a polyacrylamide gel [15]. Mouse immunization, production of the stable hybridoma clones and samples of the ascite liquid, MA isotyping, and EIA were carried out according to the standard procedures [16, 17]. MAs were isolated from the ascite liquid by precipitation with the semisaturated solution of ammonium sulfate, followed by the affinity chromatography on the protein-G-Sepharose according to the manufacturer’s instructions. The immunoblotting
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CT46(–)
Del10(–)
NT30(+)
19K(+)
16K(+)
Del3(–) Del3(+) 28K-1(–) 28K-1(+) 28K-2(–) 28K-2(+) 28K-3(–) 28K-3(+) GFP(+)(w/o start)
GFP(–)(w stop)
SphI-MP(+)
EcoRI-MP(–)
EcoRI-GFP(+)(w/o start) T T T T G A A T T C C C T A G A G T G A G C A A G G G C G A G G A G C T G
P4
P5
P6
P7
P8
P9 P10 P11 P12 P13 P14 P15 P16 P17
P18
P19
P20
P21
PstI
ApaI
EcoRI
EcoRI
T T T TGAAT TCTCCTCCAGGGG T AAACA T CG T CGG
SphI
T T T TGCATGC T TGC T C TAGT TGT TAAAGG
PstI
T T T T C TGCAGT TAC T TGTACAGC T CGT CCATGCC
BamHI
C TGGGTGGT TATAGC TGTGTAGTAAGAT CC GGAT C T TAC TACACAGC TATAACCACCCAG CCAAACCGGCGT T CACC T C TGACAATGAC CAGAGGTGAACGCCGGT T TGGT CGT CACGGG ACCAGACACACCGTGACGACCAAACCGGC GGT CGT CACGGTGTGT C TGGTGGACAAAAGG GC TGC TGTGTACC T T T TGT CCACCAGACACAC GGACAAAAGGTACACAGCAGC TGCAAAG T T T TGGAT CCATAAGAACGGGGCCCAGAGTGAGCAAGGGGGAGGAGC TGT
BamHI
AAGGAT CCAT CAAAAACGT C TGGCAAGT T T TA
BamHI
AAGGAT CCAT CGAAAGAGCCGACGAGGCCAC T
BamHI
AAGGAT CCGTAAAGAGTGT TATGTGT T CC
PstI
AT CGACC TGCAGT TAT CCGT C T C T CAC T T TGTAAT C T T
PstI
AT CGACC TGCAGT TAT T TGCGGACAT CAC T C T T T T T T CCGG
PstI
ATGCAC TGCAGT TAAT CAT CAT CGAT TAAAT TAT T C
BamHI-MP(–)
P24
BamHI
ApaI
AT CGGGAT CCAAACGAAT CCGAT T CGGCGACAG
NcoI
AT CGCCATGGC T C TAGT TGT TAAAGGAAAAGTG
BamHI * The restriction sites of the corresponding endonucleases are underlined.
NcoI-MP(+)
BamHI-GFP(–)(w/o stop) T T T T G G A T C C T A T T C T T G C G G G C C C T C G C T T G T A C A G C T C G T C C A T G C C
P23
P22
CT11(–)
P3
GT CGACC TGCAGT TAAAACGAAT CCGAT T CGGCGAC
BamHI
PstI-pQE-MP(–)
P2
3')
AAGGAT CCACGGAC TAGT TGT TAAAGGAAAAGTGAAT
Oligonucleotide structure* (5'
BamHI-pQE-MP(+)
Name
P1
Designation
Table 3. Sequences of the oligonucleotide primers used in this study
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was carried out according to the standard technique described in [18]. The full-size MPr (positive control), the deletion variants of MPr, and the recombinant PME (negative control) were separated by PAGE in 12% PAG and transferred onto the PVDF membrane, in order to determine the MPr epitope bound to the concrete MA in immunoblotting. The ascite liquid at the dilution of 1 : 10000 was used for the further coloring by antibodies. The typical pattern of immunoblotting is presented in Fig. 2. The results of epitope mapping of MA are given in Table 1 and Fig. 3. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 04-48490-a. REFERENCES 1. Heinlein, M. and Epel, B.L., Int. Rev. Cytol., 2004, vol. 235, pp. 93–164. 2. Tomenius, K., Clapham, D., and Meshi, T., Virology, 1987, vol. 160, pp. 363–371. 3. Heinlein, M., Padgett, H.S., Gens, J.S., Pickard, B.G., Casper, S.J., Epel, B.L., and Beachy, R.N., Cell, 1998, vol. 10, pp. 1107–1120. 4. Reachel, C. and Beachy, R.N., Proc. Natl. Acad. Sci. USA, 1998, vol. 95, pp. 11 169–11 174. 5. McLean, B.G., Zupan, J., and Zambryski, P.C., Plant Cell, 1995, vol. 7, pp. 2101–2114.
6. Deom, C.M., Oliver, M.J., and Beachy, R.N., Science, 1987, vol. 237, pp. 389–394. 7. Wolf, S., Deom, C.M., Beachy, R.N., and Lucas, W.J., Science, 1989, vol. 246, pp. 377–379. 8. Citovsky, V., McLean, B.G., Zupan, J.R., and Zambryski, P., Genes Dev., 1993, vol. 7, pp. 397–411. 9. Atabekov, J.G. and Dorokhov, Yu.L., Adv. Virus Res., 1984, vol. 29, pp. 313–364. 10. Citovsky, V., Knorr, D., Schuster, G., and Zambryski, P., Cell, 1990, vol. 60, pp. 637–647. 11. Citovsky, V., Wong, M.L., Shaw, A.L., Venkataram Prasad, B.V., and Zambryski, P., Plant Cell, 1992, vol. 4, pp. 397–411. 12. Brill, L.M., Nunn, R.S., Kahn, T.W., Yeager, M., and Beachy, R.N., Proc. Natl. Acad. Sci. USA, 2000, vol. 97, pp. 7112–7117. 13. Ivanov, K.I., Ivanov, P.A., Timofeeva, E.K., Efimov, V.A., and Dorokhov, Yu.L., Bioorg. Khim., 1994, vol. 20, pp. 751–757. 14. Hochuli, E., Dobeli, H., and Schacher, A., J. Chromatogr., 1987, vol. 411, pp. 177–184. 15. Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685. 16. Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1988. 17. Sukhacheva, E.A., Novikov, V.K., and Ambrosova, S.M., Bioorg. Khim., 1994, vol. 20, pp. 1060–1069. 18. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1989.
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