Photochemistry and Photobiology Vol. 56, NO. 5, pp. 717-723, 1992

Printed in Great Britain. All rights reserved

0031-8655192 $05.00+0.00 Copyright @ 1992 Pergamon Press Ltd

STRUCTURAL STUDIES ON THE PHOTORECEPTOR PHYTOCHROME: REEVALUATION OF THE EPITOPE FOR MONOCLONAL ANTIBODY Z-3B1 J. BONENBERGER~, R. SCHENDEL', H. A. W. SCHNEIDER-POETSCH~ and W.

RUDIGERI*

LBotanisches Institut der Universitat Miinchen, Menzingerstrasse 67, 8000 Miinchen 19, Germany and *Botanisches Institut, Universitat zu Koln, Gyrhofstrasse, 5000 Koln, Germany (Received 27 December 1991; accepted 6 May 1992)

Abstract-The photoreceptor phytochrome is widely distributed in the plant kingdom from angiosperms to ferns, mosses and algae. The epitope for the monoclonal antibody Z-3B1 which exhibits wide-ranging cross-reactivity with phytochromes from higher and lower plants was mapped by the combination of several methods: by Western blot with proteolytic fragments of known localization, by sequence comparison of phytochromes from various plants, and by production of overlapping fusion proteins. The only sequence which is common to all positively-reacting fusion proteins is the sequence A-830 to R-859. This sequence must contain the Z-3B1 epitope. The best candidate is suggested to be the T-cell antigenic sequence K-Y-V/I-E-A/C-L-L-T (=K-848 to T-855). The significance of the highly conserved epitope in all phytochromes is discussed.

INTRODUCTION

The photoreceptor phytochrome mediates many aspects of light-regulated growth and development in higher plants (Shropshire and Mohr, 1983; Furuya, 1987). Phytochrome or phytochrome-like proteins have been detected in a large number of plants ranging from angiosperms to ferns, mosses and algae (review: Rudiger and Thummler, 1991). Monoclonal antibodies were used for this purpose in several laboratories (Cordonnier et al., 1983, 1986; Schneider-Poetsch, 1988; Lopez-Figueroa et al., 1989, 1990). Two monoclonal antibodies exhibited wide-ranging cross-reactivity, viz. Pea-25 raised against pea phytochrome (Cordonnier et al., 1986) and Z-3B1 raised against maize phytochrome (Schneider-Poetsch et al., 1988). The epitope for monoclonal antibody Pea-25 was mapped at amino acid residues 765-771 (Cordonnier, 1989; Thompson et af., 1989) whereas only a preliminary report on the epitope for Z-3B1 is available (Grimm et al., 1986) suggesting a localization in the chromophorecarrying domain. We report here on a more precise localization of the epitope for monoclonal antibody Z-3B1. MATERIALS AND METHODS

Phytochrome (124 kDa) was isolated from 3.5-day-old etiolated oat seedlings (Avena sativa L . , cv. Pirol, Baywa, Miinchen, Germany) as previously described (Grimm and Riidiger, 1986). Partial proteolysis with trypsin (from pig) and endoproteinase Glu-C (from Staphylococcus aureus V8) was achieved according to previously described procedures (Grimm et al., 1986, 1988). All enzymes were from Boehringer, Mannheim, Germany. The sodium dodecylsulfate-polyacrylamide gel elec-

'To whom correspondence should be addressed.

trophoresis was carried out according to Laemmli (1970). Proteins were transferred to nitrocellulose (SS BA 85, Schleicher & Schiill) by the method of Towbin et al. (1979). Antibody-antigen complexes on Western blots were detected as described by Blake et al. (1984). Molecular weight standards were obtained from Sigma Chemical Co., St. Louis, MO. Cloning in the expression vector hgtll and sequencing of two C-terminal phytochrome fragments (Seq 1 found in a cDNA library from Selaginella rnartensii Spring, and Seq 2 from a Zea mays L. library) was performed as described by Schneider-Poetsch and Braun (1991). Generation of subclones for Avena phytochrome. The full-length cDNA (HM4.1,3.7 kb) for Avena phytochrome was derived from a hgtll library (Bonenberger, 1989; Thiimmler et al., 1992). Digestion with Eco RI yielded a 3.5 kb insert which was ligated into pBluescript KS+. Subclones were obtained either by digestion with different restriction enzymes or by Exo III/Mung Bean nuclease deletions. Digestion with Pvu I1 and Xba I yielded a 1167 bp-fragment which was isolated by preparative gel electrophoresis and religated into the frame of lac Zu of pBluescript KS+. This construction was used to generate a deletion series of the phytochrome coding region. According to the protocols of Stratagene (Bluescript Exo/Mung Bean nuclease DNA Sequencing System, Instruction Manual), the conditions of the ExolMung Bean nuclease digestion were chosen so that the products should differ about 100 bp in length. In addition, two cDNA fragments were generated by digestion with Hinc II/Xba I and Msp IlEco R I, respectively. Both fragments were ligated as mentioned above into pBluescript KS+. With all constructions, the fusion junction between lac Zu and cDNA inserts of phytochrome was verified by the dideoxy chain termination method of DNA sequencing (Biggins et al., 1983) using T7 DNA polymerase (Pharmacia). Finally, the inserts of these subclones were ligated into the Kpn I site of the pMAL-c polylinker (New England Biolabs, Protein Fusion and Purification System, No. 800) in order to increase the expression of the phytochrome polypeptides, now fused to the maltose-binding protein encoded by this vector. Expression of fusion proteins. Expression was carried out in E. coli strain DH 5a. Three mL of LB medium (Maniatis et al., 1982) containing 100 kg/mL ampicillin

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were inoculated with 0.3 mL of an overnight culture of transformed bacteria and incubated for 30 min at 37°C. Expression of the fusion proteins was induced by adding IPTG to give a final concentration of 1 mM. Cultures were incubated for further 2.5 h and cells were harvested by centrifugation for 1-2 min at 13OOO rpm in a microcentrifuge. Cells were resuspended in 100 p,L SDS sample buffer (Laemmli, 1970) and lysed by boiling for 5 min. 5-10 )rL of the lysate were loaded to the subsequent 10% SDSPAGE. RESULTS AND DISCUSSION

The previously suggested epitope mapping for monoclonal antibody Z-3B1 (Grimm et al., 1986) was deduced from the positive reaction with proteolytic fragments from oat phytochrome (23.5, 31,39, 59, 114, 116, 118 kDa) which were supposed to map within the N-terminal, chromophore-binding domain. However, finding two C-terminal phytochrome fragments which reacted with antibody Z3B1 in expression cDNA libraries cast doubts on this assignment. One fragment was derived from Selaginella phytochrome (Seq 1) (Schneider-Poetsch and Braun, 1991), the other (Seq 2) from Zea phytochrome (Schneider-Poetsch et al., 1991). According to the sequence, the fragments stretch 406 and 484 amino acids directly from the C-terminus; alignment with the amino acid sequence of oat phytochrome (Hershey et a l . , 1985) yields the localization from residues 730 to 1129 (including 3 gaps). It is very unlikely that the clones for these two fragments had been picked up on the basis of some unspecific reaction with the antibody. The Z-3B1 epitope should therefore be localized within this sequence. A careful reinvestigation of the reaction of Z-3B1 with proteolytic phytochrome fragments supported this conclusion. Trypsin digestion (Fig. 1) was performed here with less enzyme (0.06%) than the amount (0.1-1%) used originally (Grimm et al., 1986). In addition to fragments smaller than 60 kDa which were originally described (Grimm et al., 1986), bands at 63-64, 70-72 and 81-83 kDa are now present. These bands, which can be stained with polyclonal antibodies, cannot be stained with Z-3B1 (Fig. 1). A strong reaction of Z-3B1 is found with the bands at 55 kDa (presumed sequence R596/E-597 to R-1093, see Grimm ef al., 1986) and at 39 kDa. For the latter band, only a sequence V66 to R-426 was described by Grimm et al. (1986). According to Lagarias and Mercurio (1985), another

AB

kDa

124 114

CD

4

--o

81-83 4

-

70-724 64-62 4 58 55

39

-

Figure 1. Immunoblot analysis of proteolytic fragments from etiolated oat phytochrome. Digestion was performed with 0.06% trypsin (wt/wt) at 4°C for 17 h and stopped by adding SDS-sample buffer and heating. Sample load was 370ng protein per lane. Peptides were separated on a 11% polyacrylamide gel. Lanes A (phytochrome in the Pfr form) and B (Pr form) were immunostained with rabbit polyclonal antibodies directed against 124 kDa phytochrome from etiolated oat. Lanes C (Pfr form) and D (Pr form) were immunostained with monoclonal antibody Z-3B1.

39-kDa polypeptide arises from cleavage at the 81-83 kDa site; it should comprise amino acid residues 754-1129 (C-terminus). This second fragment might have been present in the experiment of Grimm et al. (1986) but was not detected by microsequencing. Digestion with endoproteinase-Glu-C (not shown) revealed that the previously described fragments at 66, 60,58 and 40 kDa which all comprise the chromophore region (Grimm et al., 1988) are also not detected by antibody Z-3B1. The missing reaction of antibody Z-3B1 with chromophore-con-

Figure 2. (opposite) Alignment of phytochrome sequences which contain the Z-3B1 epitope: Seq 1 (Schneider-Poetsch and Braun, 1991), Seq 2, Zea (Christensen and Quail 1989), Avena (Hershey et al., 1985), Oryza (Kay et al., 1989), Pbum (Sato, 1988), Cucurbita (Sharrock et al., 1986),Arabidopsb (Sharrock and Quail, 1989). Identical amino acid residues are indicated by colons. Antigenic epitopes predicted by the method of Hopp and Woods (1981) are double underlined. Possible T-cell antigenic sites predicted by the method of Rothbard and Taylor (1988) are underlined. In the line consensus, positions which contain identical amino acids in these sequences (*) or conservative amino acid exchanges (%) are marked. Sequences of 5 or more such marked amino acids are underlined. The numbering of amino acids includes the initiating M so that the base numbers (Fig. 3) can directly be compared with the amino acid numbers.

Epitope mapping of anti-phytochrome antibody

Residue No. Seq 1 Seq 2 z.mays A . sativa 0.sativa P.sativum C.pePQ A.thaliana Consensus Residue No. Seq 1 Seq 2 .?.mays A. sativa 0.sativa P .sativum c . ?epo A.thaliana Consensus Residue No, Seq 1 Seq 2 2 .mays A . sat iva 0 .sat iva P. sat ivum C.pepo A . thaliana Consensus Residue No. Seq 1 Seq 2 2.mays A.sativa 0.sativa P.sativum C.pepo A. thaliana Consensus Residue No. Seq 1

719

73 0 LVG

F:A::M:VH:L:::::::VE::::::IH::::::::::::::::W:::::A::T::T F:A::M:VH:L:::::::VE::::::IH::S.::::::::::::W:::::A::T::T F:A::I:A::T:::::::*E::::: : : : : : :Q:: : : : : :T::::W:C:: :A::I: :T F:A: :I::::M:::::: :LE::::::.::::::::::: :W::::::: :A::T F:,H:L::::T::: :::::E. . . . . . I : :. .. .. .. .. .. .. ... .. :S::: :T::::W:T::::::S::T ******g ** ****** * ** * *** ** +*x * ** * * *******X 790

820

850

880

GWRRE V IQMMYCRLKG MIVWSAADGQDTEKFPFAFFDRQG : : H : DY'K?LCEEssNAs :L : : s FVRLC:II:::LA:EEA::A:IG::::D: 7 ::H:D::VD:::L::V:NSSNAS:L::SK::FVRLC::I:::LA:EEA::AS:G::::N: ::N:D:::D:::L::V:DSSNAS:P::NR::FVSLCVLI:::LA:EE:::A::G::::S: ::H:D::IN:::L::V:DSTNAS:LV:NK::FVSLC:LI:::~:DE:::A::S::::N: ::K::::MD:::L::V::T::SC::::N:E:~:G::::K:MT:LE:::V::G::S:K: :.S::::ID:::L::V::VHKSC::::N:E:FVNLG::::N:MC:::P::AS:G:LA:N: :&:: : :ID:..L::V::T:KSC::.:N:E:EVNLG:: : :N:VTS::PD:VS:: : :T:G: ** *%** **x**t.x* * i* *** x x * * * X * * ***? GS ITGVFCFUAEL~QALTVQLKELGLHPPE : :I:C:: s :W::::::I:VP:DD::H::H::Q:S:QT::RR::AFSYM ::::C::SV":D:W::::::I:VP:DD::H::H::Q:S:QT:QR:::AFSYMRHA ::X:C::S:NRKENEG:L:::::::I:V::H:::H.. ::Q:S:QTS:KR::AFSYMRHA : :I:C::SV":D:V: : : : : : :IQVP:H:: :HI:!i :Q:SQQN::T:: :AYSYMRHA ::::C::SVS:KI::::LV:::::::QL::P::::::HI::LS:QT::KR::V:TYMKRQ M:::C::CVN:IL:KD:AV::F::::QLP:H::::::NI::LC:QT::KR:RA::YIKRQ '":C::CVS:KL:RK:W::,..::QL::H:.....H** :LA:RT:VKR::A:AYIKRQ **i*x**x * g******X, x * x**'** **

KWEAWLT

r* 95 0 LSEDQKQWETGTVC& y p + DMDLESIED-m :D:E:MRQ:RVADN: . . . :L:QDN:T:KSPU C:D::

920

IKNPLY :DK:: s :NK::S:~YS:ETLKS:G:N:E:~Q:RV:DN:HR:LN:::A:L:QDN:T:KSSC:D:: :N:::S:MLYS:KALKN:::N:E:MKQIHV:DN:HH::N:::A:L:QD::TEKSSC:D:E :N:::S:KLYS:KALKN:G:N:E:MKE:WADS:HR:LN:::S:L:QD:QlWSSC:D:E :R:::A::V:SSKML:G:::ETE::RI:N:SSQ:QR:LS:::::S::DG:I:--: ::D:E :Q:::S::I:S:R:L:R:E:GVE::ELLR:SGL:Q:::S:V::ES:XDK:I:--:F~D:E :R:::S::::::KMI:G:E:GPE:RRIL:S~:Q::LS:::::S:::::IE--:C:D:E * *+ * * * X* * * *%*** * * x t.x *x*x 980

1010

1040

1070

Sea 2

2.mays

A . sativa

0.sativa P.sativum C.?epo A . thal iana Consensus

Residue No. Seq 1 Seq. 2 2.mays A . sativa 0 . sativa

P.sativum

c . pepo

A , thaliana

Consensus Residue No. Seq I Seq 2 2 . mays A . sat iva 0.sativa P . sat ivum C.P e p A . thaliana Consensus

~ S E N W - V G I ~ R L G ( X V H W k & E ~ I J G V G L P E E L V Q -R ~RG K:S:AGGSMISS:LTK--NSI:ENL:LIDF:L: :K:Q:A:V:A:ILSQ:YGEDN:EQS K:S:AGGSVDISS:LTK--NSI:ENL:LIDF:.::K:R:A:V:A:ILS:YEEDNKE SE K: s : VGGsvEIss : L m --NsI: E m :LID: : i::K :Q : L:v :A::m$: : EEDNKESSE K:S:VGGSVEISCSLTK--NSI:ENL:LID::L::K:Q:K:V:AD:LSQ:YEDDNKEQSD NS::NGGQWIAASLTK--EQ::KS::LVN::LS:::G:S:V::AALNQ::G"V-LESE SYA::GGQLTISTD:TK--NQ::KS::LV:::::::YA:G:I::S:LN:::GSEE-DASE N::::GGQLT:SASLRK--DQ::RS::LAN::I:L::T:A:I::F:LNQ::GTEE-DVSE %*

x

x

%*

* * * * *x*

x*x

1140

T

*

'LVSLELPLAQRDDAGSVKFQASS 1LTA::AA:PSAAGH 1LTA::AA:PSAVGR 1ITA::AS:PTAMGQ 1L:V::AS:PAK 1L:V::AA:HKLKG 1ITT::AA:HKSRTT IITA: :AA:NK

xx

Fig. 2-fegeiitl opposite.

** *

%

J . BONENBERGER et al.

720

2 -381

. . pMcPP

1 1582 - 1

pMc25

1582

pMcl!

1582

pMcPX

1582

2411

. . . 2509 . .

.* .. 2558 2351 1 - 1

pMc HX

. .

2769

. . '. ... ...

2769'

+

2609'.

pMc 1C

I I

Pvu II

3138.

25.00

I

Hirkll Mspl

X$I

3000

3500

. .

3615

EC~RI

Figure 3. Scheme representing the cDNA subclones used for epitope mapping. Starting material was the full-length cDNA HM4.1 (Bonenberger, 1989; Thiimmler et al., 1992) which is presented at the bottom together with the restriction sites used for subcloning. Restriction with Msp I and Eco RI, Hinc I1 and Xba I, or Pvu I1 and Xba I yielded the subclones pMcl4, pMcHX or pMcPX, respectively. Digestion of pMcPX with ExoIIUMung Bean nuclease yielded the subclones pMcll, pMc2S and pMcPP. Initiating and terminating bases were verified by sequencing, except for those marked with an asterisk. The resulting fusion proteins (see Fig. 4) showed a positive reaction with mAB Z-3B1 except for the products of clones pMcPP and pMc25 as indicated to the right.

taining fragments, especially with the 83 kDa-fragment which stretches from the N-terminus to presumably residue K-753 (Grimm et al., 1988), excluded the localization of its epitope in the Nterminal half of phytochrome. Antibody Z-3B1 reacts with phytochrome from a great number of different plant species (SchneiderPoetsch et af., 1988). This means that the epitope should be highly conserved. Since a sequential epitope consists of about 6 amino acids, one can assume that an identical sequence of at least S-6 amino acid residues is required for a common epitope. We have therefore compared the new sequences of phytochrome fragments (Seq 1 and Seq 2) with the sequences of phytochrome from various plants (Fig. 2). When we consider all sequences which have 5 or more amino acid residues identical or closely related with the new sequences (see * or YO in the line consensus, Fig. 2), only 9 candidates for the epitope of antibody Z-3B1 remain. These are underlined in the line consensus (Fig. 2). The antigenic epitopes of Seq 1 predicted by the method of Hopp and Woods (1981) (sequences double underlined in Fig. 2) are not present in any other of the investigated phytochrome sequences; these can therefore be excluded as candidates for the Z-3B1 epitope. The method of Rothbard and Taylor (1988) predicts a great number of T-cell antigenic sites (underlined in Fig. 2). If T-cell antigenic sites are also immunogenic for B-cells, we consider one of these sequences a candidate for the epitope of Z-3B1. In order to check this hypothetical assignment for the Z-3B1 epitope experimentally, a series of overlapping fusion proteins were generated. Briefly, a full-length cDNA clone for Avena phytochrome (Bonenberger, 1989) was subcloned after restriction with several restriction enzymes and by further deletion of subclones with exonuclease as indicated

in Fig. 3. The 5'- and 3'-terminal base sequences of the inserts were controlled by sequencing in order to verify the identity of the N- and C-terminal amino acids and also the correctness of the reading frame. By subcloning of the inserts into the plasmid pMALc, the desired sequences were fused to that of the maltose binding protein. Expression of the fusion proteins in transformed E. coli was tested by SDSPAGE of total proteins which were extracted after induction with IPTG. Staining of the gels with coomassie blue (not shown) revealed strong bands at the expected size for all clones except for clone pMc25. Identification of the fusion proteins was achieved by immunostaining of the blots with a polyclonal antiserum directed against oat phytochrome [Fig. 4(A)]. The strong bands detected by staining with coomassie blue gave also a strong immunoreaction. In addition, a narrow protein band was immunostained at the expected position for the fusion of clone pMc25. Immunostaining with monoclonal antibody Z-3B1 [Fig. 4(B)] gave a positive reaction with fusion proteins from clones pMcl4, pMcHX, pMcPX and pMcll but a negative reaction with those of clones pMc25 and pMcPP. The only region which is common to all positive clones is that from base 2489 to base 2558 (see Fig. 3); this corresponds to amino acid residues A-830 to R-859. The epitope for Z-3B1 must be localized within this sequence. The previously discussed Tcell antigenic site (K-848 to T-855) is contained within this sequence. We suggest that this might be the epitope proper; this assignment needs experimental verification. The antigenicity of this site must be low. N o antiphytochrome antiserum was reported to possess a trace of cross-reactivity with this monoclonal antibody. Despite the low antigenicity, however, the recognized region must be at the surface of native phytochrome: Z-3B1 reacts

Epitope mapping of anti-phytochrome antibody

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8 kDa

- 68 -121

- 45 - 36 - 29 - 24 - 20 -

1 2 3 4 5 6 7 8

1 2 3 1 5 6 7 8

kDa

- 121 -

68

-

-

45

-

36

-

-

29

-

-

24

-

20 -

Figure 4. Immunoblot assay of fusion proteins expressed in E. coli by subclones according to Fig. 3. The same products were applied to the 4 gels (A-D). Lane 1 contains authentic phytochrome isolated from etiolated oat seedlings. Equal amounts of E. coli lysate were applied to lanes 2-8, except for lane 6 which contains a 10-fold amount of lysate. Lane 2: clone pMcl4 (expected size of the fusion protein 76.8 kDa); lane 3: pMcHX (56.5 kDa); lane 4: pMcPX (84.8 kDa); lane 5 : pMcll (77.8 kDa); lane 6: pMc25 (76 kDa); lane 7: pMcPP (72.4 kDa); lane 8: non-recombinant pMc. Immunostaining was performed with rabbit antibodies to phytochrome (A), with non-immune rabbit immunoglobulins (C), with monoclonal antibody Z-3B1 (B), and with non-immune mouse immunoglobulins (D). Arrowheads indicate the positions of fusion proteins. Positions of molecular mass markers are indicated in the middle.

721

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with phytochrome (Pr and Pfr) in its native conformation (Schneider-Poetsch et al., 1989). The epitope t o which Z-3B1 binds belongs to the highly conserved sequence regions of phytochrome (Schneider-Poetsch et a l . , 1988) comparable with the conservation of the sequence P-766 t o E-772 which was identified as an epitope for monoclonal antibodies Pea-25, Pea-2 and Oat-15 (Cordonnier, 1989; Thompson et al., 1989). T h e antibody Z-3B1 had been raised against phytochrome from etiolated maize seedlings; the epitope should therefore be contained in “type I” phytochrome. Cross-reactivity of Z-3B1 has also been found, however, with phytochrome from green plants including several ferns, mosses, a liverwort (Schneider-Poetsch et al., 1989, 1988) and several macroalgae (L6pez-Figueroa et a l . , 1989, 1990). T h e sequence of Selaginella phytochrome (see Fig. 2) is more closely related t o phyB than to phyA (Schneider-Poetsch and Braun, 1991). It is probable therefore that the Z-3B1 epitope is not restricted to “type I” phytochrome. This assumption is corroborated by a computer search with the partial sequence G-832 t o S-855 by the FASTA program in the data bank of Martinsried (MIPS): all phytochrome sequences (including phyB) are found with scores >90, phyC with a score of 75, but no other protein sequences with scores >60. It should be noted, however, that extracts from several plants which should contain phytochrome did not cross-react with Z-3B1 (see Schneider-Poetsch et al., 1988). The epitope is close t o a region of about 250 amino acids which share a striking homology with bacterial sensor proteins known t o transduce environmental signals by conformational alteration to the transcriptional machinery (Schneider-Poetsch and Braun, 1991; Schneider-Poetsch et al., 1991). It remains to be shown whether the conserved sequence to which Z-3B1 binds is part of the signal transducing part of the phytochrome molecule. Acknowledgements-We thank Dr. J. G. Sgouros, Martinsried, for help with the sequence data bank. W . Rudiger thanks the Deutsche Forschungsgemeinschaft, Bonn, for support. Hj. A. W . Schneider-Poetsch thanks the Volkswagenstiftung, Hannover. REFERENCES

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