evidence for internal sequence homologies in leguminosae lectins: phylogenetical implications

Biochemical Systematics and Ecology, Vol. 18, No. 1, pp. 29-37, 1990. 0305-1978/90 $3.00+ 0.00 Printed in Great Britain. © 1990 Pergamon Press plc.

Evidence for Internal Sequence Homologies in Leguminosae Lectins: Phylogenetical Implications

PIERRE ROUGI ~ and JEAN-LOUP RISLER* Laboratoire de Biologie Cellulaire, Facult~ des Sciences Pharmaceutiques, 35 chemin des Mara~chers, 31 062 Toulouse Cedex,

France; *Centre de Gbn6tique Moleculaire du C.N.R.S., 91 19B Gif sur Yvette Cedex, France

Key Word Index--Vicieae; Leguminosae; lectins; internal homologies; gene duplication; structure-function relationships.

Abstract--Three stretches of homologous amino acid sequences were recognized along the complete amino acid sequences of two-chain and single-chain Leguminosae lectins. These stretches, of about 28 residues each, were, respectively, located at both ends and in the middle of the polypeptide chain, and exhibited a significant proportion of homologous (20-40%) residues. In addition, they occurred in regions which are essentially buried in nature and contain a high proportion of I]-sheet structures. These findings suggest that genes coding for lectins probably arose by duplication of a common ancestral gene followed by limited divergence. However, as a result of such a common ancestry of Leguminosae lectins, the three- dimensional fold of their polypeptide chains was much better conserved than their constituent amino acids. This could explain why, besides a common structure-function relationship, lectins exhibit different fine sugar specificities.

Introduction Lectins from the Leguminosae have been exten- sively studied over the past decade [1-3], and the results gathered on their structure and chemical composition clearly show that they correspond to a homogeneous group of closely related proteins [4]. However, legume lectins have been classified into two distinct groups [5], according to the number of their constituent polypeptide chains:

(1) single-chain lectins are tetramers (mol. wt approx. 100,000) built up from the non-covalent association of identical monomers made of a single chain or subunit (tool. wt approx. 25,000) of about 240 amino acid residues. Soybean lectin (SBA), Dolichos biflorus lectin (DbA) or Con A from Canavalia ensiformis DC. seeds are good examples of such a structure.

(2) Two-chain lectins are dimers (mol. wt approx. 50,000) made of two identical monomers, each of them corresponding to the non- covalent association of a heavy subunit or I]- chain (mol. wt approx. 20,000) of about 180 residues, with a light subunit or 0{ chain (mol. wt approx. 5000) or 50--55 residues. Lectins from seeds of garden pea (PsA), broad bean (favin),

(Received 21 September 1989)

lentil (LcA) or Lathyrus species--all members of the tribe Vicieae--belong to this group.

In addition, single-chain lectins are inhibited by D-galactose and its derivatives while two- chain lectins are inhibited by D-glucose, D-mannose and their derivatives. However, some dis- crepancies were shown to occur, e.g. Con A or Dioclea grandiflora DC. lectin are tetrameric lectins inhibited by D-glucose and g-mannose while the sainfoin (Onobrychis viciifolia L.) lectin is a dimeric lectin also inhibited by D-glucose and D- mannose. Morever, such a clear-cut distinction between single- and two-chain lectins does not always appear as relevant since the recent discovery of Lathyrus lectins which are typical single-chain lectins, e.g. Lathyrus sphaericus Retz [6] and Lathyrus nissolia L. [7] lectins. In this respect, seeds of Vicia cracca L. have to be men- tioned because they contain both single- and two-chain lectins of different sugar specificities [8]. The amino acid sequences of lectins from the members of the Vicieae appear to be closely related, whatever the number of their constituent subunits (Fig. 1). However, typical single-chain lectins from other Leguminosae tribes exhibit less closely related amino acid sequences. In addition, a circular permutation has to be made between Con A and other single- or two-chain

29

30 PIERRE ROUGE AND JEAN-LOUP RISLER

1.LoLl : 'I~I'PS~SITKFGPD

2.LoLII : ..............

3 .LaphL: ...... L .... S--

4 .LartL: ..............

5 .LcicL: ...... L .......

6. PsA ....... L .... S--

7 .Favin: TD-I ..... P--R--

8 .LcA : ........... S--

9 .LnLI : ...... L .... SA-

i0. LnLII : ...... L .... SA-

20 30 40 50

QQNLIFQGDG FITKERLTLTKAVRNTVGRALYSSP I HI WDSK'PGN -p ...........................................

.............. S-K-L ..... K .....................

.............................................

..............................................

............... K ....... K ................ RE---

-P ..... -(3 .............. K ......... L ....... E---

............... K ..... VSKE-G ...... T ...... RD-V-

.............. DK-L ....................... Q---

.............. DK-L ....................... Q---

ii. LsphL: TE ........ P-TDQPSSPKFVSG-P ...... NA-S-DGK-I--E-KQ ......... A ...... R---K

60 70 80 90 i00 ii0 120

1. VANFVPSHT FVIDAPNSY NVAEX3FTFFIAPVDTKPQTGGGYLGVF NSKOYDKTSQTVAVEF~PFYNTAWD

2. - ...... A ...............................................................

3. ----I ........ N .......................................... K ........... A---

4. - ......................................................................

5. - ........... N .................................. V .................. A---

6. - ........... N .................................. AE .... T ............ A---

7. --D-T-T-I ....... G ............................ -G ...... A ............ A---

8 . - ..... NGSQVFRES--G ............................ Y-G-E ................. A---

9. - ........... N ......................................... K ............ A---

i0. - ........... N ......................................... K ........... A---

If. --D-TA .... Y-R ---DSQV .............. Q-RGD--L ..... REE--P-IH ......... H-QP--

130 140 150 160 170 180 l((x ) i0

i. PSNGDRH IGI DVNS I KS I ~I"KS WKLQNGKEA NVVIAFNAATNVLTVS LTYPN E'PS YTLNEVVPLKEF 2. - ..................................... G ............................

3. ---K ............. V ..................... E .......... S- A ......... A--DV

4. - ................................................... A ..............

5. ---RE ....................... V ....................... V ............ DV

6. ---R ............. V .......... E ....................... SLEEEN V ..... SD--S--DV

7. ----K ....... ~ ..... S .... N .... E--H-A-S .... r .... S-T-L--- L-G---S ...... DV

8. ---KE ............ V ..... N ....

9. - ................ V .................................. . ...... SV ............ DV

i0. - ................ V .................................. . ...... SV ........ A--DV

ii. -DY I---V-I ..... RI-RP-NPHYDTYSIAY--YK .... E-D-TV .... S ...... RDYA--R---D--QI

20 30 40 50

i. VPEWVRIG FSA'I~AEFAAHEVISWFFHSELAGTSSSN

2 . - ......................... Y-N---SV .....

3. - ..... V .................. S-Q---S---G--

4. - ........................ S ............

5. - ........................ S .... -GE--A-KQ

6. - ................ Y ........ S ..... S ..... KQ

7. - ............... Y-T ...... T-L---T-P-N

8. - ........................ S-N-Q-GH--K-

9. - ..... V .................. S ......... A-KQ

io. - ........................ s ..... D .....

ii. - ..... V-L--S-ATYYS .... Y--S ..... G .......

FIG. 1. COMPARISON OF THE AMINO ACID SEQUENCES OF TWO CHAIN AND SINGLE CHAIN SEED LECTINS OF THE VICIEAE TRIBE [LoLI/LoLII: isolectins from Lathyrus ochrus (L.) DC.; LaphL, lectin from L. aphaca L; LartL, lectin from L. art/culatus L.; LcicL, lectln from L. cicera DC.; PsA, lectin from Pisum sativum L.; Favin, lectin from Vicia faba L.; LcA, lectin from Lens culinaris Medik.; LnLI/LnLII, isolectins from L. nissolia L.; LsphL, lectin from L. sphaericus Retz]. Residues identical to LoLl chosen as model were indicated by dashes (----) and gaps were introduced to maximize the homologies. Points (....) indicate that polypeptide chains are uninterrupted.

lect ins in o rde r to m a x i m i z e the h o m o l o g i e s tha t relate all these lect ins. Such a pe rmu ta t i on , r e p o r t e d by C u n n i n g h a m e t aL [9], w a s inter- p re ted on the basis o f a c o m p l e x m e c h a n i s m of dup l i ca t i on and fus ion o f an ancestra l gene c o d i n g for the p o l y p e p t i d e chain o f lect ins, wh i ch

cou ld exp la in the st ructura l re lat ion occu r r i ng b e t w e e n Con A (or D. g r a n d i f l o r a DC. lectin) and o the r lect ins [10]. Recent e x p e r i m e n t s on the b io- syn thes is o f Con A [11-13] have s h o w n tha t post - t rans la t iona l even ts o f the p recurso r chain of Con A cou ld be so le ly respons ib le fo r such an un-

INTERNAL SEQUENCE HOMOLOGIES IN LEGUMINOSAE LECTINS 31

expected relation. Another model based on the conformational homologies existing between all these lectins led Olsen [14] to suggest that all legume lectins are composed of three equivalent structural domains. We present here preliminary results on the occurrence of internal homologous stretches along the amino acid sequences of legume lectins, which corroborate the hypothetic model proposed by Olsen [14].

Results and Discussion Three stretches of homologous sequences could be identified along the amino acid sequences of the two Lathyrus ochrus isolectins (LoLl and LoLII) by comparing these sequences to them- selves with the DotPIot program [15], using proper window and stringency values. Both the Dayhoff [16] and the Risler [17] matrices gave very similar results but the latter was chosen because it allows an easier recognition of the stretches of repeated sequences. By using the BestFit program [15], these stretches were shown to correspond, respectively, to residues 50-77, 149-176 and 188-215, for which the percentages of homology vary from 21.4 to 39.3% (Fig. 2). The corresponding stretches are easily recognizable along the amino acid sequences of other two- chain and single-chain lectins, but we have to take into account the circular permutation that

relates Con A or D. grandiflora lectin to other Leguminosae lectins [9] in order to localize these stretches along their amino acid sequences (Fig. 3). The stretches 50-77, 149--176 and 188-215 of LoLI/LoLII correspond, respectively, to stretches 179-205, 37-64 and 76-103 of Con A. The alignment of these three stretches from various single- and two-chain lectins gives an overall percentage of homology ranging from 54 to 57.5%, depending on the grouping [16-18] of exchangeable amino acids (Fig. 4A-C). These results suggest that a gene duplication process was involved in the emergence of the genes coding for lectins, even though the original amino acid sequence similarities of the resulting repeated units were subsequently restricted to less extended stretches of homologous sequences. The search into the MIPS data bank of other sequences homologous to these three lectin stretches with the Isearch program [19], shows that Leguminosae lectins always give the highest score values.

By reference to pea lectin [20], two-chain lectin monomers are made of two major and one minor antiparallel J3-sheet structures whose strands are interconnected by a variety of turns and loops to form a 13-barrel structure• The two major antiparallel sheets consist of two halves of, respectively, six (back face of the molecule) and

FIG. 2. ALIGNMENT OF THE THREE RESIDUE STRETCHES OF THE L. OCHRUSAMINO ACID SEQUENCE RECOGNIZED AS HOMOLOGOUS (stretches 50-77, 149-176 and 188-215). Residues considered as homologous according to the Risler's scoring matrix [17] (score values ranging from 1.9 to 2.2) were boxed from one to another sequence.

(i) (181) (i) (53)

LoLI i ............. ~ ................ I m I m m m ~ .... 234

SBA i ............... ~ .............. n ~ m ....... 253

Con A 1 .............. ~ ............... ~ . . . . . . . m 237

(123) (237) (i) (122)

FIG 3 OVERLAPPING OF IHE AMINO ACID SEQUENCES OF TYPICAL TWOCHAIN (LoLl isolect[n I of L ochrus) AND SINGLECHAIN (SBA from Gly cine max) LECTINS WITH THAT OF CON A, ACCORDING TO THE CIRCULAR PERMUTATION REPORTED BY CUNNINGHAM ETAL, [9]. Stretches of homologous sequence are indicated by strengthened lines.

32 PIERRE ROUG~: AND JEAN-LOUP RISLER

LoLI 50

149

188

LaphL 50

149

188

PsA 50

149

194

Favin 51

150

190

LnLI 50

149

188

DbA 54

151

191

SBA 57

151

191

Con A 179

37

76

A

seven (front face of the molecule) strands. We refer to back and front faces of the pea lectin monomer by analogy with the organization of the Con A dimer [21]. Altogether, with an additional minor sheet of three strands which lies on the left border of the molecule and which appears as the continuation of strands from the two major sheets, these two halves form the framework of the molecule which looks like a flattened bell shaped dome. Such a very stiff

FIG. 4A

77

176

215

77

176

215

77

176

221

78

177

217

77

176

215

81

178

218

84

178

218

205

64

103

structure also occurs in other two-chain and single-chain lectins, e.g. in ravin [22], L. ochrus (L) DC. lectin [23] and Con A [21, 24]. The three stretches of the lectin sequences which were recognized as being homologous correspond to regions of both the back and front faces of the molecule where several (6/13) 13-sheet strands are located (Fig. 5). They essentially coincide with buried regions of the polypeptide chains of lectins (Fig. 6) and, therefore, these homologous


LoLl

LaphL

PsA

Favin

LnLI

DbA

SBA

Con A

B 5O

149

188

5O

149

188

5O

149

194

51

150

190

5O

149

188

54

151

191

57

151

191

179

37

76

stretches contain a significant proportion (7/12) of those residues which were shown to build up the hydrophobic cavity of the Con A monomer [25] and which are believed to play a major role in the proper folding of lectins [23]. This hydrophobic portion of lectin molecules could be responsible for the binding of various plant hormones and derivatives on lectins [26, 27].

Owing to the fact that the homologous stretches mainly coincide with well conserved

FIG. 4B

77

176

215

77

176

215

77

176

221

78

177

217

77

176

215

81

178

218

84

178

218

2O5

64

103

regions of the amino acid sequences of both two-chain and single-chain lectins, one can suppose that their occurrence in legume lectins could have something to do with the very conserved structure-function relationship of these molecules [28]. Interestingly, each of these stretches falls into one of the three regions of the amino acid sequences of legume lectins previously recognized as domains by Olsen [14], on the basis of structural comparisons between

C LoLI 5O

149

188

LaphL 50

149

188

PsA 50

149

194

Favin 51

150

190

ImLI 50

149

188

DbA 54

151

191

SBA 57

151

191

Con A 179

37

76

77

!

]

E

I

S

I

T

N

I

T

S

I

T

S

V

T

S

34 PIERRE ROUG~: AND JEAN-LOUP RISLER

215

176

215

77

176

176

221

78

177

217

77

176

215

77

81

218

84

178

218

2O5

64

103

178

FIG. 4. COMPARISON OF THE THREE STRETCHES OF HOMOLOGOUS SEQUENCE FROM VARIOUS "FVVO-CHAIN (LoLl, isolectin I from L. ochrus; LaphL, lectin from L. aphaca; PsA, lectin from R sativum; Favin, lectin from ~Z faba) AND SINGLE-CHAIN LECTINS (LnLI, isolectin I from L. nlssolia; DbA, lectin from D. biflorus; SBA, lectin from Glycine max; Con A, lectin from C. ensiformis). Homologous residues were boxed according to the grouping of homologous amino acids of (A) Risler et al. [17], (B) Dayhoff et al. [16] and (C) Swanson [18]. In order to discriminate between various groups of homologous residues which could occur at a given position, those homologous residues which were found to be the most largely distributed among two or more sequence stretches belonging to a single or different lectins were taken into account. Score values ranging from 1.9 to 2.2 in the Risler's scoring matrix were used to define homologous residues, since they correspond to the highest values allowing a convenient alignment of related amino acid sequences with the 8estFit program [15].

single- and two-chain lectins. According to this author, all legume lectins arose from the com- bination of three (I, II, III) structural domains corresponding, respectively, to residues 1-69 (I), 70-122 (Ill) and 123-237 (111) of Con A and, due to

the circular permutation between Con A and other lectins, to residues 112-185 (I), 186-240 (11) and 1-115 (111) of other single-chain lectins, and to residues 112-185 (I, p-chain), 1-50/55 (11, ~.-chain) and 1-115 (111, j]-chain) of two-chain lectins. Our


5O (~) < . . . . . . . . . . . >

62-72

77

149 176 (~) < . . . . . > < . . . . . . . . > < . . . . . . . . >

147-151 160-167 172-179

188 215 o• ..........................

(o(i) < ........ > < .......... > (o(.28)

188-195 206-215

FIG. 5, COINCIDENCE BETWEEN THE THREE HOMOLOGOUS STRET- CHES OF THE PEA LECTIN (PsA) AND THE LOCALIZATION OF ~-SHEET STRANDS (INDICATED BY ARROWS) ALONG THE HEAVY (~) AND LIGHT (c() SUBUNITS OF THE LECTIN. Six strands over a total of 13 strands for the whole lectin molecule fall into the three stretches.

h o m o l o g o u s s t re tches exac t l y co inc ide w i t h d o m a i n s I (stretch 149-176), II (stretch 188-215) and III (stretch 50-77) . As in m a n y pro te ins , the t h r e e - d i m e n s i o n a l fo ld o f lect ins w a s m u c h be t te r conse rved than the i r cons t i t uen t a m i n o ac ids w h i c h cou ld exp la in that , bes ides a struc- t u r e - f u n c t i o n re la t ionsh ip c o m m o n to all l e g u m e lect ins, d iscre te d i f fe rences in the i r a m i n o acid sequences m i g h t s u p p o r t the d i sc repanc ies o b s e r v e d in the i r f ine sugar spec i f ic i t ies [29, 30].

A l t h o u g h it is t e m p t i n g to specu la te a b o u t the ro le o f gene dup l i ca t i on in the e m e r g e n c e in p ro te ins o f n e w st ructura l fea tu res a d a p t e d to spec i f ic f unc t i ons [31, 32], the occu r rence o f in terna l sequence h o m o l o g i e s in l e g u m e lect ins w h i c h s e e m s to be co r re la ted w i t h the i r structure, re in forces the n o w w i d e l y a c c e p t e d op in i on tha t p lan t lect ins c o r r e s p o n d to ve ry we l l conse rved mo lecu les . Accord ing ly , t hey a p p e a r m o r e and m o r e as va luab le p h y l o g e n e t i c ma rke rs [33], a carefu l s t udy o f wh i ch , associ - a ted w i t h the c o m p a r i s o n of the i r a m i n o acid or gene sequences , cou ld he lp us to i m p r o v e bo th the de l i nea t i on and p h y l o g e n e t i c re la t ionsh ips o f va r ious lec t in -con ta in ing taxa w i t h i n the L e g u m i - nosae. In add i t i on , such a d e g r e e o f s t ructura l conse rva t i on sugges ts tha t lect ins shou ld p lay an i m p o r t a n t ro le in l e g u m e plants, p e r h a p s in re la t ion to s y m b i o s i s w i t h R h i z o b i a [34] o r w i t h de fens i ve m e c h a n i s m s aga ins t p lan t pa thogen i c bac te r ia or fung i [35].

Experimental The complete amino acid sequences of single- and two-chain legume lectins were taken from the following references: L. ochrus (L) DC. isolectins [36, 37]; L. aphaca L., L. articulatus L.

and L. cicera L. lectins [38, 39]; L. nissolia L. lectin [6]; L. sphaericus Retz lectin [7]; Pisum sativum L. lectin [40, 41 ]; Vicia faba L. lectin [42, 43]; Lens culinaris Medik. lectin [10, 44]; C. ensiformis DC. lectin [45]; D. grandiflora DC. lectin [46]; Glycine max L. Merr. lectin [47]; Onobrychis viciifolia L. lectin [48]; D. biflorus L. lectin [49]; Phaseolus vulgaris L. lectin subunits [50].

Various computerized programs from the Wisconsin Genetics Computer Group [15] were used to search for internal homologies in lectins: we used the Compare and DotPIot programs, and the BestFit program based on the algorithm of Smith and Waterman [51], in conjunction with the Dayhoff's [16] and the structural superposition [17] scoring matrices. The Isearch program [19] of the PIR (National Biomedical Research Foundation, Washington D.C., U.S.A.) was used to compare the recognized homologous stretches to other proteins of the MIPS (M6nich, West Germany) data bank for protein sequences. The hydropathic profiles of lectins were predicted according to [52], and assessed by comparison with other profiles resulting from flexibility [53] and accessibility to solvent [54].

References 1. Etzler, M. E. (1985) Ann. Rev. Pl. Physiol. 36, 209. 2. Lis, H. and Sharon, N. (1986) Ann. Rev. Biochem. 53, 35. 3. Van Driessche, E. (1988) in Advances in Lectin Research

(Franz, H., ed.) VoI. 1, p. 73. Springer, Berlin. 4. Roug~, P., Ranfaing, P., P~re, D., Richardson, M., Yarwood,

A. and Sousa-Cavada, B. (1986) in Lathyrus and Lathyrism (Kaul, A. K. and Combes, D., eds) p. 273. Third World Medical Research Foundation, New York.

5. Foriers, A., De Neve, R. and Strosberg, A. D. (1979) Physiol. Veg. 17, 597.

6. Richardson, M., Yarwood, A. and Rouge, P. (1987) Fedn Eur. Biochem. Socs Lett. 216, 145.

7. Yarwood, A., Richardson, M., Morphet, B., Westby, M., P~re, D. and Roug~, P. (1988) Phytochemistry27, 1719.

8. R(~diger, H. (1977) Eur. J. Biochem. 78, 317. 9. Cunningham, B. A., Hemperly, J. J., Hopp, T. P. and Edel-

man, G. M (1979) Proc. Natn. Acad. Sci. U.S.A. 76, 3218. 10. Foriers, A., Lebrun, E., Van Rapenbusch, R., De Neve, R.

and Strosberg, A. D. (1981) J. Biol. Chem. 256, 5550. 11. Carrington, D. M., Auffret, A. and Hanke, D. E. (1985)

Nature 313, 64. 12. Bowles, D. J., Marcus, S. E., Pappin, D. J. C., Findlay, J. B.

C., Eliopoulos, E., Maycox, P. R. and Burgess, J. (1986) J. Cell. Biol. 102, 1284.

13. Chrispeels, M. J., Hartl, Ph.M., Sturm, A. and Faye, L. (1986) J. Biol. Chem. 261, 10021.

14. Olsen, K. W. (1983) Biochim. Biophys. Acta 743, 212. 15. Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucl.

Acids Res. 12, 387. 16. Dayhoff, M. O., Eck, R. V. and Park, C. M. (1972) in Atlas of

Protein Sequence and Structure, Vol. 5, p. 89, Natl. Biomed. Res. Foundation, Washington D.C.

17. Risler, J. L., Delorme, M. O., Delacroix, H. and Henaut, A. (1988) J. Mol. Biol. 204, 1019.

18. Swanson, R. (1984) Bull. Math. Biol. 46, 623. 19. Dayhoff, M. O., Barker, W. C. and Hunt, L. T. (1983) Methods

Enzymol. 91, 524. 20. Einspahr, H., Parks, E. H., Suguna, K., Subramanian, E. and

Suddath, F. L. (1986) J. Biol. Chem. 261, 16518. 21. Reeke Jr, G. N., Becket, J. W. and Edelman, G. M. (1975) J.

Biol. Chem. 250, 1525.

36 PIERRE ROUG~ AND JEAN-LOUP RISLER

(a)

40

30

20

~ ,o

---~---i

!

,o ,o ~o I [ IOO I:~O

Residue number

/b/s° t __;__~ , i ,ol~ .A , , ,,

1// °tt " "! t/ - I 0

I

- 2 0 ? , I | / I ',

- 3 0 ~ I I V

| I I '-;- . . . . ,J I 0 ~'0 40 60 80 I00

,4'0

I I I

I J . I l i

i I I I I I I I t I

leo 180

r- . . . . . "i r 3 I r i r I r I

' t' I J I

11 Ull i l l r

- - - L . . . . J

200 ;~20

~--~-- F-~-- i i i i

A IAt' ' , I 1 I I

ii I ', . . . . . A _

HI : , /i1 ( I

IV' i/IVI It ' '

r'[v I! ' ' ,,

I I L - T . . . . . . . . . . ~ I I:~0 140 laO 180 ;)00 ;=20 240

Residue number

FIG. 6. REPRESENTATIVE SURFACE PROFILES FOR (A) LaphL (L. aphaca lectin) AND (B) SBA (Glyclne max lectin). Surface profiles were calculated with the HPLC values of Parker et al. [52]. The lower line represents the mean protein hydrophilicity value and the upper line represents the 25% cut off value described in [52]. Peaks over the upper line correspond to the most exposed sequence stretches. The three dashed boxes numbered 1, 2 and 3 correspond, respectively, to the three homologous stretches 50-77 (1), 149-176 (2) and 188-215 (3) and essentially coincide with peaks located under the lower line which are predicted to be buried.

22. Reeke Jr, G. N. and Becker, J. W. (1986) Science234, 1108. 23. Bourne, Y., Cambillau, Ch., Boisseau, C., Richardson, M.

and Rouge, P. (1989) in Lectins, Biology, Biochemistry, Clinical Biochemistry (Bog-Hansen, T. C. and Freed, D. L. J., eds) Vol. 6, p. 265. Sigma Chemical Co., St. Louis, Missouri, U.S.A.

24. Hardman, K. D. and Ainsworth, C. F. (1972) Biochemistry11, 4910.

25. Becket, J. W., Reeke Jr, G. N., Wang, J. L., Cunningham, B.

A. and Edelman, G. M. (1975) J. Biol. Chem. 250, 1513. 26. Edelman, G. M. and Wang, J. L. (1978) J. Biol. Chem. 2 5 3 ,

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evidence for internal sequence homologies in leguminosae lectins: phylogenetical implications

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