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ELISA measurement of immunochemical cross-reactions among Lathyrus lectins as an assessment of their phylogenetical relationship
BiochemicalSysternaticsand Ecology,Vol. 15, No. 3, pp. 349-353, 1987. Printed in Great Britain.
0305-1978/87 $3.00+0.00 ¢) 1987PergamonJournals Ltd.
ELISA Measurement of Immunochemical Cross-reactions among Lathyrus Lectins as an Assessment of their Phylogenetical Relationship P. ROUGI~, M. L. GARCIA, C. BOISSEAU and H. CAUSSE Laboratoire de Biologie cellulaire, Facultb des Sciences Pharmaceutiques, Universit(~ Paul Sabatier, 35 chemin des Maralchers, 31 062 Toulouse, France
Key Word Index--Lathyrus; Vicieae; lectins; immunochemical cross-reactions; phylogenetical relationships. Abstract--The immunochemical cross-reactions among Lathyrus and other Vicieae lectins have been quantified by using an ELISA (enzyme-linked immunosorbent assay) technique, in order to assess their phylogenetical relationship. The data are consistent with those arising from the comparison of both the amino acid compositions of the lectins and the amino acid sequences of their light and heavy subunits. They confirm that two-chain lectins from the tribe of Vicieae are phylogenetically closely related.
Introduction Amino acid or nucleotide sequence analyses are required for unambiguous evidence of structural and evolutionary relationships among proteins and, subsequently, these relationships can be used to assess the evolutionary relatedness among the organisms from which proteins are obtained. This is especially true for legume lectins but, until now, very little information is available concerning the complete amino acid or nucleotide sequences of lectins [1]. Immunochemical studies on lectins offer a quite different but powerful tool for determining relationships between legume lectins, provided that highly specific antibodies against lectins are readily available. However, all the existing immunochemical techniques are not suitable for such a purpose and the sufficiently specific and sensitive ones have to be preferred in order to avoid misleading or confusing results. Thus, using the very simple Ouchterlony double diffusion method [2], Hankins et al. [3] and Howard et al. [4] were able to detect cross-reactions between single-chain lectins belonging to various Leguminosae tribes whereas, in other hands [5], the same technique failed to detect these antigenic identities. Similarly, Ouchterlony double diffusion showed that single- and two(Received 4 July 1986) 349
chain legume lectins were antigenically unrelated [5-8], although extensive amino acid sequence homologies exist when these lectins are compared [9, 10] and we possess now compelling evidence that legume lectins are structurally and evolutionarily related proteins [9-11]. Recently, a more sophisticated ELISA technique has been successfully used by Michaelsen and Kolberg [12] to detect the common antigenic determinants present both inside and outside the carbohydrate binding site of various single- and two-chain legume lectins. We have adapted a similar technique to quantify the cross-reactions among Lathyrus and other Vicieae lectins and we report here on our preliminary results.
Results By performing the retained indirect ELISA technique on various Lathyrus (L. aphaca, L. articulatus, L. cicera, L. gmelinii, L. hirsutus, L. ochrus, L. odoratus and L. tingitanus) and other Vicieae (Lens culinaris and Pisum sativurn) lectins, results obtained in both homologous and heterologous reactions for three different lectin concentrations (0.5, 1.0 and 3.0 lig/ml, respectively) were similar to those shown in Fig. 1, in which specific antibodies against L. aphaca and L. ar~'culatus lectins have been used. The three-point curves obtained in homologous
350
P. ROUGI~, M. L. GARCIA, C. BOISSEAU AND H. CAUSSE
.7JA41o
.5
A
A41o
B
ochrus lectin-antibodies against L. odoratus lectin, and L. odoratus lectin-antibodies against L. ochrus lectin reactions) were compared. However, despite these discrepancies, the L. tingitanus lectin appears as less related to other Lathyrus and Vicieae lectins while soybean lectin always remains very weakly related to other two-chain lectins. Discussion
In order to assess the phylogeneticat significance of the cross-reactivity percentages, they have been compared with the currently available 1 .~ amino acid sequences of the light [13-19] and heavy [20-23] subunits of various Lathyrus and Vicieae lectins (Figs 2 and 3) and the homo?- . . . . . . 7 . . . . . . . . . . . ? 0.5 1,0 3.0 0.5 1.0 3,0 logous regions of soybean lectin [24]. Very few LECTIN CONCENTRATION t4g/ml differences exist among the amino acid FIG. 1. ELISA RESULTS OBTAINED IN BOTH HOMOLOGOUS AND sequences of both the light and heavy subunits HETEROLOGOUS REACTIONS BETWEEN DIFFERENT CONCENTRA- fro the Lathyrus and Vicieae lectins, whereas the TIONS OF LATHYRUS AND VlCIEAE LECTINS AND IgG CORhomologous regions of soybean lectin exhibit RESPONDING RESPECTIVELY TO LATHYRUS ARTICULATUS (A) AND LATHYRUS APHACA (B) LECTINS. The following symbols have been less extensive similarity. Although this comused for lectins: Lathyrus aphaca (~r), L. articulatus (O), L. cicera (~), parison agrees with our data and is therefore in L. gmelinii (~), L. hirsutus (ill), L. ochrus (&), L. odoratus (IB), L. favour of the phylogenetical significance of the tingitanus (0), Lens culinaris (~), Pisum sativum (R) and G/ycine max (- - © - -). cross-reactivity percentages calculated from the ELISA curves, it does not take into account howreactions exhibited higher absorbance values ever, sequence changes occurring in the than those measured in heterologous reactions antigenic determinants of the lectin subunits and this was true for all the assayed Lathyrus which are indeed the sole structural features and related Vicieae (lentil and pea) lectins. Soy- that could affect the ELISA results. To overcome bean lectin from Glycine max, which belongs to this difficulty, the method of Hopp and Woods the tribe Glycineae, did however react to a [25] was applied to the more variable light sublesser extent with antibodies against Lathyrus units from Lathyrus and Vicieae lectins in order lectins, giving three-point curves of very low and to predict the location of their antigenic deteroften just measurable absorbance values (Fig. minants. A comparison of the obtained hexa1). peptide profiles (Fig. 4) clearly shows that In order to quantify these cross-reactions beside a major antigenic determinant located among Lathyrus and other Vicieae lectins, cross- near the N-terminal end, all the Lathyrus and reactivity percentages (homology percentages) Vicieae light subunits possess another deterwere calculated from the ELISA curves at an minant that coincides with their very C-terminal antigen concentration of 1.0 p.g/ml, using the end. Soybean lectin exhibits however a quite absorbance value with the homologous antigen different pattern. Interestingly, the most as 100%, and then compared among the important changes observed among the amino assayed lectins. The cross-reactivity percent- acid sequences of Lathyrus and Vicieae lectins ages obtained in such a simple way are shown (see Fig. 2) occur in this region. It is now in Table 1. According to the data, Lathyrus and currently postulated that peptides of more than Vicieae lectins obviously correspond to very 7 amino acids which are located in flexible and closely related proteins, even though some dis- accessible regions of an intact protein [26] could crepancies occurred in those few cases where act as good antigenic determinants. According the corresponding homologous cross-reactions to the hypothesis of Olsen [27] concerning the (e.g. the comparison of values obtained with L. presence of three distinct functional domains in
351
ELISA MEASUREMENT OF CROSS-REACTIONS AMONG LATHYRUS LECTINS TABLE 1. HOMOLOGY OR CROSS-REACTIVITY PERCENTAGES (%) CALCULATED FROM ELISA CURVES Lectins from:
L.ap.
L.ar.
L.ci.
L.gm.
L.hi.
Lap. L.ar. L.ci. Lgm. L.hi. Loc. L.od. L.ti. Le.c. P.sa. G.max
100 83 74 93 70 78 52 72 93 73 13
82 100 71 82 85 86 77 28 88 60 16
51 65 100 61 78 50 47 28 94 65 21
74 45 90 100 80 41 78 29 64 92 16
65 72 71 75 100 61 80 45 50 70 13
Antibodies from: L.oc. L.od. 51 98 63 71 80 100 58 35 68 49 15
70 86 85 99 87 81 100 53 94 85 14
L.ti.
Le.c.
P.sa.
G.max
52 66 70 78 45 51 61 100 70 55 4
79 81 90 66 79 55 58 38 100 83 21
79 66 97 80 79 53 67 41 68 100 18
8 3 2 3 4 12 11 2 5 7 100
Lap., Lathyrus aphaca; L.ar., L. artfculatus; L.ci., L. cicera; L.gm., L. gmelinii; L.hi., L. hirsutus; L.oc., L. ochrus; L.od., L. odoratus; L.ti., L. tingitanus; Le.c., Lens cu/inaris; P.sa., I~'sum sativurn; G.max, Glycine max.
Con-A and other single- and two-chain lectins, light subunits from Lathyrus and Vicieae lectins correspond to domain II where the C-terminal end is coveniently exposed in order to fulfil the requirements needed for suitable determinants. Moreover, the comparison indicates that determinants are similarly located on the light subunits of the Lathyrus and Vicieae lectins, into a region which only differs by limited amino acid sequence changes. Accordingly, only subtle differences of these determinant sequences are likely to account for the limited immunochemical diversity observed among the Vicieae lectins, in such a way that only monoclonal antibodies Vicieae
:
Lat.och. c( I : Lat.och. o(2 : Lat.aphaca : Lat.act. : Lat.cic. o( 1 : Lat.cic. o<2 : Lat.hir. o< I : Lat.hir. o<2 : Lat.odoratus : Lat. sativus : Lat.tingitanus:
I0 20 30 40 EI~YTLNEV V P L K E F V P E W VP.IGFSATIU AEFAAHEVLS W F F H S E L A G T -Y-N--SV--v---g-o---s-A----A--DV -S A - - V - - -DV -S GEV - - ---DV -S GEV - - --DV--S,, G--V - - .DV--S ~ V - - DV--s--~ V - - .DV.--S DG-V I -,- D V - -S EE-
Lens culinaris : V - - -DV-~"s--um sativum : V SD- - S - - D V - Glycineae
~
could completely discriminate among these very closely related species. Thus, the comparison of both lectin amino acid sequences and antigenic determinants strongly supports the phylogenetical significance of the crossreactivity percentages calculated from the ELISA curves. On the other hand, L. ting#anus has been shown to occupy a position rather far from other species in the phylogenetic tree based upon the amino acid composition of the light subunits of the Lathyrus lectins (unpublished results) and constructed according to the method of Cornish-Bowden [28], that agrees with our data showing that L. #'ng#anus lectin
S-N-0-GH- - - -
--y.___
-s
50 SSSN .
-G--A-KQ -A-0 -A-KQ -A-O
-C~0K -A-KOS -A-K0 -K-
-s-- --KQ
:
max
17--
: R--NI-SD- -D--TSL
---A--
LDIPGg-~---- - S - A - N - P H A --NINP
/SH~"
FIG. 2. COMPARISON OF THE AMINO ACID SEQUENCES OF THE LIGHT SUBUNITS OF SOME LATHYRUS LECTINS. Lat.nch., L. ochrus; I,at.art, L. articulatus; Lat.cic., L. cicera; Lat.hir., L. hirsutua Data concerning lentil, pea and soybean iectins have been included, g, Gap introduced to maximize the homologies.
352
P. ROUGI~,M. L. GARCIA,C. BOISSEAUAND H. CAUSSE Vicieae : 10 20 30 4O ~ 1 : T E T F S F S I T KFGPDQONLI FOGI~YTI"gKE P~LTLTKAVRN TggggggVGRALYSSP iff 2 : Pg gggggg . . . . . . : .... L-- --S g S - K-L . . . . K- - g g g g g g ( 3 )Lat. aphaca : .... L g gggggg ( 4 )Lat .cicera
( 1 )Lat.och. ( 2)Lat.och.
( 5 )Lens culin. ( 6 )Pisum sat. Glycineae
: : ....
S L-- --S
~g-- G------VSKE -ggggggG-------Tg-- K - - - - - - K - -g_ggggg . . . . . .
:
( 7 ~31ycine max
:AEg-V---IgN --V-K-P-M- LG--AIVTSSG K - O - N - ~
GTPKPSSL----T-T-
( i -T8~7 50 60 70 80 90 IO0 I 10 (I): IHIWDSKTGN VANFVTSFTgF VIDAPNSYNV ADGFTFFIAP VDTKFQTSC~ YLGVFgNSKDY DKTSQ ( 2 ): A--g -g ( 3 ): (4): ( 5 ): - - - - - R D - V -
--
I----g-
--N-gDN --NGSQV F R E S - - C ~ - -
-g K -g--V . . . . -Y-G-E . . . .
-
( 6 ): - - - - R E g --N(7): -----KE--S --S-AA--Ng- TFY--DTKRL ---LA--L-- I . . . .
(1): (2): (3): (4): (5): (6): (7):
-g--AE- - - T HA- ---L-g-ENES ggGD-
120 130 140 150 160 170 ] TVAVE FDTFYNTAWD PSNGDRHIGI DVNSIKSINT KSWKLONGKE ANVVIAFNAA TNVLTVSLTY PN C~ ---A----K-A--- ---I~E-A-- ---KE-
V. . . .
-A---
R-Sg--
-V -V. . . .
--I%
-P-ggP----
E
S-
V-V
N---JR--K-
N--E-
T--D-A-N-V
( SLEEEN ) -K-L-TYD-S -SL-VA--V- -SO
FIG. 3. COMPARISON OF THE AMINO ACID SEQUENCES OF THE HEAVY SUBUNITS OF SOME LATHYRUS (Lat.och., L. ochrus) LECTINS. Sequences of lentil (Lens culin.: Lens culinaris) and pea (Pisum sat., Pisum sativum) and soybean lectin have been included, g, Gap introduced to maximize the homologies.
seems less immunologically related to other Lathyrus lectins (see Table 1). Threfore, it seems reasonable to use the cross-reactivity percentages calculated from ELISA curves to estimate the phylogenetical relatedness among Lathyrus and Vicieae species. The preliminary results reported here indicates that species from the Vicieae tribe have to be considered as very evolutionarily closely related species. Results obtained with soybean lectin also indicate that Vicieae seem poorly related to other singlechain lectin-containing Leguminosae tribes. Experimental P/ant material. Seeds from Lathyrus species (L aphaca L., L articu/atus L, L. cicera L, L. gmelinii Fritsch, L. hirsutus L., L. ochrus (L.) DC., L. odoratus L., L. tingitanus L.), lentil (Lens cu/inaris Medik., verte du Puy), pea (Pisum sativum L., petit provencal nain) and soybean (G/ycine max (L.) Merr., cv. Hodgson), were harvested from plants cultivated under field conditions. Isolation of/ectins. Seed flour from lentil, pea and Lathyrus species was soaked in 0.05 M Tris (pH 7.6), 0.15 M NaCI and extracted overnight under continuous stirring. After centrifugation, proteins were ppted at 90% (NH4)2SO4 and the resulting ppte was dissolved in Tris buffered saline (TBS) and
dialysed against TBS. The dialysed protein soln was filtered through a Sephadex G1O0 column equilibrated with TBS and most proteins were washed out until the A at 280 nm remained constant and lower than 0.01. The lectins were eluted with TBS containing 0.1 M glucose, ppted at 90% (NH4)2SO4, dialysed against TBS and stored frozen at - 3 0 ° until used. Soybean seed lectin was prepared according to Allen and Neuberger [29]. The purity of lectins was checked by disc PAGE at basic pH [30] and protein fractions were fixed and stained according to ref. [31]. Protein determination. Total soluble protein was estimated colorimetrically [32], using bovine serum albumin (BSA, Sigma) as standard. Preparation of antibodies. Antibodies against lectins were raised in rabbits in the presence of Freund's complete adjuvant, according to an immunization schedule previously described [33]. Immunoglobulins (IgG) were separated from other serum proteins according to Fourcart et aL [34] and thoroughly used instead of complete immune sera. Enzyme-linked immunosorbent assay (ELISA). Microtitre polystyrene plates (Linbro, SI MVC 96, Flow Laboratories) were used and incubations were performed in polystyrene boxes containing wet filter paper. Lectin solns (150 p.I of 0.1 M Na2CO3 (pH 9.6) containing respectively 0.5, 1.0 and 3.0 p.g lectin/ml), were placed in wells and allowed to adsorb at 4° during 12 h. The wells were washed 3 times by flooding with WPBS (pH 7.4) (1 I of WPBS contains 0.408 g KH2PO4, 1.22 g K~HPO4, 8.77 g NaCI and 0.05% Tween 20). A soln of BSA (250
ELISA MEASUREMENT OF CROSS-REACTIONSAMONG LATHYRUSLECTINS 2-
0-
A
O-
B
0-
C
o.1 o-t o-t o-1 o-t o-1 0-t
D E Y
F G H I J
2-
K F
I"~
I
SEQUENCE NUMBER
1 10 20 30 40 50 FIG. 4. HEXAPEPTIDE PROFILESOF LATHYRUSAND VICIEAE LECTINS CALCULATED ACCORDING TO REF. [25] WITH ~/INDOWS' OF 6 RESIDUES. A, B, C, D, E, F, G, H, I and J correspond respectively to the light subunits of Lathyrus aphaca (A), L. ar~'culatus (B), L cicera 0tl (C), L. cicera ~.2 (D), L. ochrus 0~1 (E), L. ochrus 0r.2 (F), L. odoratus (G), L. sarivus (H), Lens culinaris (I) and Pisum sativum (J). The homologous region (185-241) of soybean (Glycine max) lectin has been included (K). Arrows indicate the emplacement of predicted antigenic determinants.
pJ) containing 10 mg BSA/ml of 0.1 M Na2CO3 (pH 9.6) was added and, after an incubation of 2 h at 37", the wells were washed 3 times by flooding with WPBS. A soln (150 t~1)of IgG (5 p,g/ml) in PBS (pH 7.6) (1 I of PBS contains 1.44 g Na=HPO4, 0.2 g KH2PO4, 0.2 g KCI, 8.0 g NaCI, 0.05% Tween 20 and 0.02% NAN3) was added and, after an incubation of 2 h at 37=, the wells were washed 3 times by flooding with WPBS. A soln (150 p,I) of alkaline phosphatase-labelled goat anti-rabbit IgG (Jackson ImmunoResearch) 1/2000 diluted in PBS was added and the wells were washed 3 times after an incubation of 2 h at 3T. A soln (150 I11) of p-nitrophenyl phosphate (Sigma, 1 mg/ml of 0.05 M Na2CO3 (pH 9.8)) was added. The reaction was stopped after a 30 min incubation at 37' by adding 50 p,I 4.0 M NaOH and the A at 410 nm was recorded using a Multiskan Titertek spectrophotometer (Flow Laboratories).
References 1. Etzler, M. E. (1985) Annu. Re~. Plant Physiol. 36, 209. 2. Ouchterlony, O. (1949) Acta Path. Microbiol. Scand. 26, 507.
353
3. Hankins, C. N., Kindinger, J. L. and Shannon, L. M. (1979) Plant Physiol. 64, 104. 4. Howard, J., Kindinger, J. L. and Shannon, L. M. (1979) Arch. Biochem. Biophys. 192, 457. 5. Roug6, P. (1975) C. R. Acad. Sc~ Ser. D 280, 21. 6. Foriers, A., De Neve, R. and Strosberg, A. D. (1979) Physiol. V~g. 17, 597. 7. Roug(~, P. (1981) Biochem. Syst. Ecol. 9, 39. 8. Bauman, C. M., R~diger, H. and Strosberg, A. D. (1981) Lectins, Biology, Biochemistry, Clinical Biochemistry (BogHansen, T. C., ed.) Vol. 1, p. 93. W. de Gruyter, Berlin. 9. Strosberg, A. D., Lauwereys, M and Foriers, A. (1983) Chemical Taxonomy, Molecular Biology and Function of Plant Lec~'ns (Goldstein, I. J. and Etzler, M. E., ed.) p. 7. Alan R. Liss, New York. 10. Becker, J. W., Cunningham, B. A. and Hemperly, J. J. (1983) Chemical Taxonomy, Molecular Biology and Function of Plant Lectins (Goldstein, I. J. and Etzler, M. E., ed.) p. 31. Alan R. Liss, New York. 11. Rouge, P., Richardson, M., Chatelain, C., Yarwood, A., Sousa-Cavada, B. and P~re, D., Lectins, Biology, Bio. chemistry, Clinical Biochemistry (Beg-Hansen, T. C. and Van Driessche, E., ed.) Vol. 5. W. de Gruyter, Berlin (in press). 12. Michaelsen, T. E. and Kolberg, J. (1984) Acta Path, Microbiol. Immunol. Scand., Sect. C 92, 25. 13. Foriers, A., De Neve, R., Kanarek, L. and Strosberg, A. D. (1978) Proc, Natl Acad. Sci. U.S.A. 75, 1136. 14. Richardson, C., Behnke, W. D., Freisheim, J. H. and Blumenthal, K. M. (1978) Biochim. Biophys. Acta 537, 310. 15. Sletten, K., Kolberg, J. and Michaelsen, T. E. (1983) FEBS Letters 156, 253. 16. Sletten, K. and Kolberg, J. (1993) Hoppe-Seyler'sZ. Physiol. Chem. 364, 1047. 17. Richardson, M., Rouge, P., Sousa-Cavada, B. and Roug~, P. (1984) FEBS Letters 175, 76. 18. Souse-Cavada, B., Richardson, M., Yarwood, A., P~re, D. and Rouge, P. (1986) Phytochemistry 25, 115. 19. Yarwood, A., Richardson, M., Sousa-Cavada, B., P~re, D. and Roug~, P. (1986) Phytochemis~y25, 2109. 20. Foriers, A., Lebrun, E., Van Rapenbusch, R., De Neve, R. and Strosberg, A. D. (1981) J. Biol. Chem. 256, 5550. 21. Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C. and Spencer, D. (1983) J. Biol. Chem. 256, 9544. 22. Yarwood, A., Richardson, M., Sousa-Cavada, B. and Rouge P. (1985) FEBSLetters 184, 104. 23. Roug~, P., Richardson, M., Yarwood, A., Ranfaing, P., Sousa-Cavada, 8. and P6re, D. (1986) Colloque Lathyrus, 913 Sept. 1985, Pau, France (in press). 24. Vodkin, L. O., Rhodes, P. R. and Goldberg, R. B. (1983) Cell 34, 1023. 25. Hopp, T. P. and Woods, K. R. (1981) Prec. NatlAcad. Sc~ U.S.A. 78, 3824. 26. Welling, G. W. and Fries, H. (1985) FEBS Letters 192, 81. 27. Olsen, K. W. (1983) Biochim. Biophys. Acta 743, 212. 28. Cornish-Bowden, A. (1977) J. Theor. Biol, 65, 735. 29. Allen, A. K. and Neuberger, A. (1975) FEBS Letters 50, 362. 30. Davis, B. J. (1964) Ann. N. Y. Acad. Scl; 121, 404. 31. Chrambach, R. A., Reisfeld, R. A., Wyckoff, M. and Zaccari, J. (1967) Analy¢ Biochem. 20, 1, 32. Goa, J. (1953) Scand. J. C/in. Lab. Invest 5, 218. 33. Rouge, P. (1977) Th~se Doct. Sc. Nat. Toulouse, no. 746. 34. Fourcart, J., Saint-Blancard, J., Girot, P. and Boschetti, E. (1982) Rev. Ft. Transf Immuno-H~.,matol. 25, 7.
0305-1978/87 $3.00+0.00 ¢) 1987PergamonJournals Ltd.
ELISA Measurement of Immunochemical Cross-reactions among Lathyrus Lectins as an Assessment of their Phylogenetical Relationship P. ROUGI~, M. L. GARCIA, C. BOISSEAU and H. CAUSSE Laboratoire de Biologie cellulaire, Facultb des Sciences Pharmaceutiques, Universit(~ Paul Sabatier, 35 chemin des Maralchers, 31 062 Toulouse, France
Key Word Index--Lathyrus; Vicieae; lectins; immunochemical cross-reactions; phylogenetical relationships. Abstract--The immunochemical cross-reactions among Lathyrus and other Vicieae lectins have been quantified by using an ELISA (enzyme-linked immunosorbent assay) technique, in order to assess their phylogenetical relationship. The data are consistent with those arising from the comparison of both the amino acid compositions of the lectins and the amino acid sequences of their light and heavy subunits. They confirm that two-chain lectins from the tribe of Vicieae are phylogenetically closely related.
Introduction Amino acid or nucleotide sequence analyses are required for unambiguous evidence of structural and evolutionary relationships among proteins and, subsequently, these relationships can be used to assess the evolutionary relatedness among the organisms from which proteins are obtained. This is especially true for legume lectins but, until now, very little information is available concerning the complete amino acid or nucleotide sequences of lectins [1]. Immunochemical studies on lectins offer a quite different but powerful tool for determining relationships between legume lectins, provided that highly specific antibodies against lectins are readily available. However, all the existing immunochemical techniques are not suitable for such a purpose and the sufficiently specific and sensitive ones have to be preferred in order to avoid misleading or confusing results. Thus, using the very simple Ouchterlony double diffusion method [2], Hankins et al. [3] and Howard et al. [4] were able to detect cross-reactions between single-chain lectins belonging to various Leguminosae tribes whereas, in other hands [5], the same technique failed to detect these antigenic identities. Similarly, Ouchterlony double diffusion showed that single- and two(Received 4 July 1986) 349
chain legume lectins were antigenically unrelated [5-8], although extensive amino acid sequence homologies exist when these lectins are compared [9, 10] and we possess now compelling evidence that legume lectins are structurally and evolutionarily related proteins [9-11]. Recently, a more sophisticated ELISA technique has been successfully used by Michaelsen and Kolberg [12] to detect the common antigenic determinants present both inside and outside the carbohydrate binding site of various single- and two-chain legume lectins. We have adapted a similar technique to quantify the cross-reactions among Lathyrus and other Vicieae lectins and we report here on our preliminary results.
Results By performing the retained indirect ELISA technique on various Lathyrus (L. aphaca, L. articulatus, L. cicera, L. gmelinii, L. hirsutus, L. ochrus, L. odoratus and L. tingitanus) and other Vicieae (Lens culinaris and Pisum sativurn) lectins, results obtained in both homologous and heterologous reactions for three different lectin concentrations (0.5, 1.0 and 3.0 lig/ml, respectively) were similar to those shown in Fig. 1, in which specific antibodies against L. aphaca and L. ar~'culatus lectins have been used. The three-point curves obtained in homologous
350
P. ROUGI~, M. L. GARCIA, C. BOISSEAU AND H. CAUSSE
.7JA41o
.5
A
A41o
B
ochrus lectin-antibodies against L. odoratus lectin, and L. odoratus lectin-antibodies against L. ochrus lectin reactions) were compared. However, despite these discrepancies, the L. tingitanus lectin appears as less related to other Lathyrus and Vicieae lectins while soybean lectin always remains very weakly related to other two-chain lectins. Discussion
In order to assess the phylogeneticat significance of the cross-reactivity percentages, they have been compared with the currently available 1 .~ amino acid sequences of the light [13-19] and heavy [20-23] subunits of various Lathyrus and Vicieae lectins (Figs 2 and 3) and the homo?- . . . . . . 7 . . . . . . . . . . . ? 0.5 1,0 3.0 0.5 1.0 3,0 logous regions of soybean lectin [24]. Very few LECTIN CONCENTRATION t4g/ml differences exist among the amino acid FIG. 1. ELISA RESULTS OBTAINED IN BOTH HOMOLOGOUS AND sequences of both the light and heavy subunits HETEROLOGOUS REACTIONS BETWEEN DIFFERENT CONCENTRA- fro the Lathyrus and Vicieae lectins, whereas the TIONS OF LATHYRUS AND VlCIEAE LECTINS AND IgG CORhomologous regions of soybean lectin exhibit RESPONDING RESPECTIVELY TO LATHYRUS ARTICULATUS (A) AND LATHYRUS APHACA (B) LECTINS. The following symbols have been less extensive similarity. Although this comused for lectins: Lathyrus aphaca (~r), L. articulatus (O), L. cicera (~), parison agrees with our data and is therefore in L. gmelinii (~), L. hirsutus (ill), L. ochrus (&), L. odoratus (IB), L. favour of the phylogenetical significance of the tingitanus (0), Lens culinaris (~), Pisum sativum (R) and G/ycine max (- - © - -). cross-reactivity percentages calculated from the ELISA curves, it does not take into account howreactions exhibited higher absorbance values ever, sequence changes occurring in the than those measured in heterologous reactions antigenic determinants of the lectin subunits and this was true for all the assayed Lathyrus which are indeed the sole structural features and related Vicieae (lentil and pea) lectins. Soy- that could affect the ELISA results. To overcome bean lectin from Glycine max, which belongs to this difficulty, the method of Hopp and Woods the tribe Glycineae, did however react to a [25] was applied to the more variable light sublesser extent with antibodies against Lathyrus units from Lathyrus and Vicieae lectins in order lectins, giving three-point curves of very low and to predict the location of their antigenic deteroften just measurable absorbance values (Fig. minants. A comparison of the obtained hexa1). peptide profiles (Fig. 4) clearly shows that In order to quantify these cross-reactions beside a major antigenic determinant located among Lathyrus and other Vicieae lectins, cross- near the N-terminal end, all the Lathyrus and reactivity percentages (homology percentages) Vicieae light subunits possess another deterwere calculated from the ELISA curves at an minant that coincides with their very C-terminal antigen concentration of 1.0 p.g/ml, using the end. Soybean lectin exhibits however a quite absorbance value with the homologous antigen different pattern. Interestingly, the most as 100%, and then compared among the important changes observed among the amino assayed lectins. The cross-reactivity percent- acid sequences of Lathyrus and Vicieae lectins ages obtained in such a simple way are shown (see Fig. 2) occur in this region. It is now in Table 1. According to the data, Lathyrus and currently postulated that peptides of more than Vicieae lectins obviously correspond to very 7 amino acids which are located in flexible and closely related proteins, even though some dis- accessible regions of an intact protein [26] could crepancies occurred in those few cases where act as good antigenic determinants. According the corresponding homologous cross-reactions to the hypothesis of Olsen [27] concerning the (e.g. the comparison of values obtained with L. presence of three distinct functional domains in
351
ELISA MEASUREMENT OF CROSS-REACTIONS AMONG LATHYRUS LECTINS TABLE 1. HOMOLOGY OR CROSS-REACTIVITY PERCENTAGES (%) CALCULATED FROM ELISA CURVES Lectins from:
L.ap.
L.ar.
L.ci.
L.gm.
L.hi.
Lap. L.ar. L.ci. Lgm. L.hi. Loc. L.od. L.ti. Le.c. P.sa. G.max
100 83 74 93 70 78 52 72 93 73 13
82 100 71 82 85 86 77 28 88 60 16
51 65 100 61 78 50 47 28 94 65 21
74 45 90 100 80 41 78 29 64 92 16
65 72 71 75 100 61 80 45 50 70 13
Antibodies from: L.oc. L.od. 51 98 63 71 80 100 58 35 68 49 15
70 86 85 99 87 81 100 53 94 85 14
L.ti.
Le.c.
P.sa.
G.max
52 66 70 78 45 51 61 100 70 55 4
79 81 90 66 79 55 58 38 100 83 21
79 66 97 80 79 53 67 41 68 100 18
8 3 2 3 4 12 11 2 5 7 100
Lap., Lathyrus aphaca; L.ar., L. artfculatus; L.ci., L. cicera; L.gm., L. gmelinii; L.hi., L. hirsutus; L.oc., L. ochrus; L.od., L. odoratus; L.ti., L. tingitanus; Le.c., Lens cu/inaris; P.sa., I~'sum sativurn; G.max, Glycine max.
Con-A and other single- and two-chain lectins, light subunits from Lathyrus and Vicieae lectins correspond to domain II where the C-terminal end is coveniently exposed in order to fulfil the requirements needed for suitable determinants. Moreover, the comparison indicates that determinants are similarly located on the light subunits of the Lathyrus and Vicieae lectins, into a region which only differs by limited amino acid sequence changes. Accordingly, only subtle differences of these determinant sequences are likely to account for the limited immunochemical diversity observed among the Vicieae lectins, in such a way that only monoclonal antibodies Vicieae
:
Lat.och. c( I : Lat.och. o(2 : Lat.aphaca : Lat.act. : Lat.cic. o( 1 : Lat.cic. o<2 : Lat.hir. o< I : Lat.hir. o<2 : Lat.odoratus : Lat. sativus : Lat.tingitanus:
I0 20 30 40 EI~YTLNEV V P L K E F V P E W VP.IGFSATIU AEFAAHEVLS W F F H S E L A G T -Y-N--SV--v---g-o---s-A----A--DV -S A - - V - - -DV -S GEV - - ---DV -S GEV - - --DV--S,, G--V - - .DV--S ~ V - - DV--s--~ V - - .DV.--S DG-V I -,- D V - -S EE-
Lens culinaris : V - - -DV-~"s--um sativum : V SD- - S - - D V - Glycineae
~
could completely discriminate among these very closely related species. Thus, the comparison of both lectin amino acid sequences and antigenic determinants strongly supports the phylogenetical significance of the crossreactivity percentages calculated from the ELISA curves. On the other hand, L. ting#anus has been shown to occupy a position rather far from other species in the phylogenetic tree based upon the amino acid composition of the light subunits of the Lathyrus lectins (unpublished results) and constructed according to the method of Cornish-Bowden [28], that agrees with our data showing that L. #'ng#anus lectin
S-N-0-GH- - - -
--y.___
-s
50 SSSN .
-G--A-KQ -A-0 -A-KQ -A-O
-C~0K -A-KOS -A-K0 -K-
-s-- --KQ
:
max
17--
: R--NI-SD- -D--TSL
---A--
LDIPGg-~---- - S - A - N - P H A --NINP
/SH~"
FIG. 2. COMPARISON OF THE AMINO ACID SEQUENCES OF THE LIGHT SUBUNITS OF SOME LATHYRUS LECTINS. Lat.nch., L. ochrus; I,at.art, L. articulatus; Lat.cic., L. cicera; Lat.hir., L. hirsutua Data concerning lentil, pea and soybean iectins have been included, g, Gap introduced to maximize the homologies.
352
P. ROUGI~,M. L. GARCIA,C. BOISSEAUAND H. CAUSSE Vicieae : 10 20 30 4O ~ 1 : T E T F S F S I T KFGPDQONLI FOGI~YTI"gKE P~LTLTKAVRN TggggggVGRALYSSP iff 2 : Pg gggggg . . . . . . : .... L-- --S g S - K-L . . . . K- - g g g g g g ( 3 )Lat. aphaca : .... L g gggggg ( 4 )Lat .cicera
( 1 )Lat.och. ( 2)Lat.och.
( 5 )Lens culin. ( 6 )Pisum sat. Glycineae
: : ....
S L-- --S
~g-- G------VSKE -ggggggG-------Tg-- K - - - - - - K - -g_ggggg . . . . . .
:
( 7 ~31ycine max
:AEg-V---IgN --V-K-P-M- LG--AIVTSSG K - O - N - ~
GTPKPSSL----T-T-
( i -T8~7 50 60 70 80 90 IO0 I 10 (I): IHIWDSKTGN VANFVTSFTgF VIDAPNSYNV ADGFTFFIAP VDTKFQTSC~ YLGVFgNSKDY DKTSQ ( 2 ): A--g -g ( 3 ): (4): ( 5 ): - - - - - R D - V -
--
I----g-
--N-gDN --NGSQV F R E S - - C ~ - -
-g K -g--V . . . . -Y-G-E . . . .
-
( 6 ): - - - - R E g --N(7): -----KE--S --S-AA--Ng- TFY--DTKRL ---LA--L-- I . . . .
(1): (2): (3): (4): (5): (6): (7):
-g--AE- - - T HA- ---L-g-ENES ggGD-
120 130 140 150 160 170 ] TVAVE FDTFYNTAWD PSNGDRHIGI DVNSIKSINT KSWKLONGKE ANVVIAFNAA TNVLTVSLTY PN C~ ---A----K-A--- ---I~E-A-- ---KE-
V. . . .
-A---
R-Sg--
-V -V. . . .
--I%
-P-ggP----
E
S-
V-V
N---JR--K-
N--E-
T--D-A-N-V
( SLEEEN ) -K-L-TYD-S -SL-VA--V- -SO
FIG. 3. COMPARISON OF THE AMINO ACID SEQUENCES OF THE HEAVY SUBUNITS OF SOME LATHYRUS (Lat.och., L. ochrus) LECTINS. Sequences of lentil (Lens culin.: Lens culinaris) and pea (Pisum sat., Pisum sativum) and soybean lectin have been included, g, Gap introduced to maximize the homologies.
seems less immunologically related to other Lathyrus lectins (see Table 1). Threfore, it seems reasonable to use the cross-reactivity percentages calculated from ELISA curves to estimate the phylogenetical relatedness among Lathyrus and Vicieae species. The preliminary results reported here indicates that species from the Vicieae tribe have to be considered as very evolutionarily closely related species. Results obtained with soybean lectin also indicate that Vicieae seem poorly related to other singlechain lectin-containing Leguminosae tribes. Experimental P/ant material. Seeds from Lathyrus species (L aphaca L., L articu/atus L, L. cicera L, L. gmelinii Fritsch, L. hirsutus L., L. ochrus (L.) DC., L. odoratus L., L. tingitanus L.), lentil (Lens cu/inaris Medik., verte du Puy), pea (Pisum sativum L., petit provencal nain) and soybean (G/ycine max (L.) Merr., cv. Hodgson), were harvested from plants cultivated under field conditions. Isolation of/ectins. Seed flour from lentil, pea and Lathyrus species was soaked in 0.05 M Tris (pH 7.6), 0.15 M NaCI and extracted overnight under continuous stirring. After centrifugation, proteins were ppted at 90% (NH4)2SO4 and the resulting ppte was dissolved in Tris buffered saline (TBS) and
dialysed against TBS. The dialysed protein soln was filtered through a Sephadex G1O0 column equilibrated with TBS and most proteins were washed out until the A at 280 nm remained constant and lower than 0.01. The lectins were eluted with TBS containing 0.1 M glucose, ppted at 90% (NH4)2SO4, dialysed against TBS and stored frozen at - 3 0 ° until used. Soybean seed lectin was prepared according to Allen and Neuberger [29]. The purity of lectins was checked by disc PAGE at basic pH [30] and protein fractions were fixed and stained according to ref. [31]. Protein determination. Total soluble protein was estimated colorimetrically [32], using bovine serum albumin (BSA, Sigma) as standard. Preparation of antibodies. Antibodies against lectins were raised in rabbits in the presence of Freund's complete adjuvant, according to an immunization schedule previously described [33]. Immunoglobulins (IgG) were separated from other serum proteins according to Fourcart et aL [34] and thoroughly used instead of complete immune sera. Enzyme-linked immunosorbent assay (ELISA). Microtitre polystyrene plates (Linbro, SI MVC 96, Flow Laboratories) were used and incubations were performed in polystyrene boxes containing wet filter paper. Lectin solns (150 p.I of 0.1 M Na2CO3 (pH 9.6) containing respectively 0.5, 1.0 and 3.0 p.g lectin/ml), were placed in wells and allowed to adsorb at 4° during 12 h. The wells were washed 3 times by flooding with WPBS (pH 7.4) (1 I of WPBS contains 0.408 g KH2PO4, 1.22 g K~HPO4, 8.77 g NaCI and 0.05% Tween 20). A soln of BSA (250
ELISA MEASUREMENT OF CROSS-REACTIONSAMONG LATHYRUSLECTINS 2-
0-
A
O-
B
0-
C
o.1 o-t o-t o-1 o-t o-1 0-t
D E Y
F G H I J
2-
K F
I"~
I
SEQUENCE NUMBER
1 10 20 30 40 50 FIG. 4. HEXAPEPTIDE PROFILESOF LATHYRUSAND VICIEAE LECTINS CALCULATED ACCORDING TO REF. [25] WITH ~/INDOWS' OF 6 RESIDUES. A, B, C, D, E, F, G, H, I and J correspond respectively to the light subunits of Lathyrus aphaca (A), L. ar~'culatus (B), L cicera 0tl (C), L. cicera ~.2 (D), L. ochrus 0~1 (E), L. ochrus 0r.2 (F), L. odoratus (G), L. sarivus (H), Lens culinaris (I) and Pisum sativum (J). The homologous region (185-241) of soybean (Glycine max) lectin has been included (K). Arrows indicate the emplacement of predicted antigenic determinants.
pJ) containing 10 mg BSA/ml of 0.1 M Na2CO3 (pH 9.6) was added and, after an incubation of 2 h at 37", the wells were washed 3 times by flooding with WPBS. A soln (150 t~1)of IgG (5 p,g/ml) in PBS (pH 7.6) (1 I of PBS contains 1.44 g Na=HPO4, 0.2 g KH2PO4, 0.2 g KCI, 8.0 g NaCI, 0.05% Tween 20 and 0.02% NAN3) was added and, after an incubation of 2 h at 37=, the wells were washed 3 times by flooding with WPBS. A soln (150 p,I) of alkaline phosphatase-labelled goat anti-rabbit IgG (Jackson ImmunoResearch) 1/2000 diluted in PBS was added and the wells were washed 3 times after an incubation of 2 h at 3T. A soln (150 I11) of p-nitrophenyl phosphate (Sigma, 1 mg/ml of 0.05 M Na2CO3 (pH 9.8)) was added. The reaction was stopped after a 30 min incubation at 37' by adding 50 p,I 4.0 M NaOH and the A at 410 nm was recorded using a Multiskan Titertek spectrophotometer (Flow Laboratories).
References 1. Etzler, M. E. (1985) Annu. Re~. Plant Physiol. 36, 209. 2. Ouchterlony, O. (1949) Acta Path. Microbiol. Scand. 26, 507.
353
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