Mitogenic properties of structurally related Lathyrus lectins

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 248, No. 1, July, pp. 30-34, 1986

Mitogenic Properties of Structurally CARL A. K. BORREBAECK’ Department of Biotechnology, University *Laboratoire de Biologic Cell&ire, Received November

AND

Related Lathyrus PIERRE

Lectins

ROUGE*

of Lund, P.O. Box 124, S-221 00 Lund, Sweden, and Universitb Paul Sabatier, Toulouse, France

20,1985, and in revised form February

21,1986

The mitogenic properties of ten phylogenetically related Lathyrus lectins have been studied. Despite a close structural resemblance and similar carbohydrate specificities, the lectins exhibited significant differences in their ability to induce cell proliferation of human lymphocytes. The differences in optimal dose were in the range lo-30 times. L. ochrus (whole lectin) also had an ability to induce cellular growth 3-10 times better than that of the isolated isolectins, L. ochrus I and II, illustrating the complexity behind 0 1986 Academic press. h. the structure-function relation of lectin mitogens.

The induction of cell proliferation and differentiation is initiated at the plasma membrane level where glycoproteins act as receptors for antigens or polyclonal activators, such as lectin mitogens or lipopolysaccharides. To explore the conditions behind a lectin-induced polyclonal cellular response it is important to understand the structure-function relationship of these mitogens (1). Structurally very similar lectins have previously been shown to exhibit quite different mitogenic properties probably due to minor differences in the carbohydrate specificity (2). Furthermore, the ability of closely related lectins to induce proliferation may also reflect differences in the induction of lymphokines necessary to obtain a cellular response (C. A. K. Borrebaeck, unpublished work). In this study we investigate the mitogenie properties of a group of phylogenetic closely related lectins belonging to the tribe Vicieae of the family Leguminosae. The lectins from the genus Lathyrus all possess an azPz structure and have recently been shown to contain striking similarities in physicochemical and biological properties (3), which were subsequently corroborated by extensive homology among the amino i To whom correspondence

acid sequences of both their light and heavy subunits (4-6). These a-D-mannose/a-Dglucose specific groups of lectins have similar carbohydrate specificity although various monosaccharides inhibited the lectin-induced hemagglutination to different degrees (3). The two Lathyrus ochrus isolectins I and II exhibited, however, identical carbohydrate specificity (7) even though our study revealed different mitogenie properties of the individual isolectins as compared to the whole Lathyrus ochrus lectin. MATERIALS

Copyright 0 1986 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

METHODS

Plant material, Seeds from Lathyrus species (L, aphaca L., L. articuiatus L., L. cicera L., L. hirsutus L., L. ochrus (L.) DC., L odoratus L., L. tin&anus L., L. vwwus (L.) Bernh.,) were harvested from plants cultivated under field conditions. Con A* was isolated (8) from Canavalia en.sifomzis DC. seed meal purchased from Serva Fine Biochemiea (Heidelberg, FRG). Isolation of lectins and isolectins. All leetins were extracted from crude seed meal using 50 mM Tris/ HCI buffer, pH 7.6, containing 0.15 M sodium chloride

’ Abbreviations used: Con A, concanavalin A, TBS, 50 mM Tris/HCl buffer, pH 7.6, containing 0.15 M NaCI; PBS, 50 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl.

should be addressed.

0003-9861/86 $3.00

AND

30

MITOGENIC

PROPERTIES

(TBS), and a fractional precipitation (30-60% (w/v)) with ammonium sulfate was subsequently performed. The precipitate was dissolved in TBS and extensively dialyzed against the same buffer. The protein extract was chromatographed on a column (70 X 5 cm) containing Sephadex G-100 equilibrated in TBS and bound lectin was eluted with 0.1 M glucose. The eluted lectins were then precipitated with 90% (w/v) ammonium sulfate. The precipitates were dissolved in TBS, dialyzed extensively against the same buffer, and the lectins were finally stored at -30°C. The separation of the two Lathyms ochrus isolectins was carried out by chromatofocusing as recently described (9). Briefly, the L ochrus lectin was applied to a PBE 94 column (30 X 1 cm) (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated with 25 mM Tris/acetic acid buffer, pH 8.4, and elution was performed using a lo-fold dilution mixture of Polybuffers 96 and 74 (30/70) adjusted to pH 5.0 using 1.0 M acetic acid. Two peaks corresponding to L. ochrus I and L ochrus II were obtained. The isolectins were precipitated by 90% (w/v) ammonium sulfate, dialyzed against distilled water, and stored at -30°C. Hemagglutination assay. Inhibition of hemagglutination was tested by a twofold serial dilution of sugars in 50 pl. PBS (50 rl), containing 37.5 pg lectin/ ml was added to each dilution. After lh incubation 200 ~1 of a 1% human erythrocyte (0 Rh+) suspension was added. The cells had previously been washed three times and resuspended in PBS. The degree of hemagglutination was estimated 12 h later. Mitogenic assay. Human peripheral blood mononuclear cells were isolated from healthy donors by Ficoll-Paque (Pharmacia Fine Chemicals) density gradient centrifugation. Activation of the lymphocytes was measured as incorporation of [methyl-3H]thymidine (5.0 Ci/mmol, Amersham International Ltd., Amersham, U.K.) into the DNA. Lymphocytes (2 X 105) in RPM1 1640, supplemented with 4 mM Lglutamine, streptomycin (50 pg/ml), penicillin (50 IU/ ml), 1% (v/v) 100X nonessential amino acids, and 10% fetal calf serum (Flow Laboratories, Inc., Ayrshire, U.K.) were incubated for 65 h in microtiter plates in the presence of each lectin. The final volume was 100 ~1 and 12 h before harvest the cells were pulsed with 1 pCi [methyl-3H]thymidine per well. When the lectininduced lymphocyte activation was performed under serum-free conditions 0.2% methyl cellulose (Methocel A4M, Dow Chemicals GmBH, FRG) was substituted for fetal calf serum. The methyl cellulose was prepared as described previously (10). RESULTS

AND

DISCUSSION

The structurally related Lathyrus lectins were tested for their mitogenic ability in culture media supplemented with fetal calf serum (10% j or methyl cellulose (0.2% j.

OF Luthyrus

LECTINS

31

These test conditions were used to investigate the interaction of the Lathyrus lectins with serum glycoproteins and how this might regulate the lymphocyte transformation (1 j. Despite the similar amino acid composition of the Lathyrus lectins (4-6) they exhibited significant differences in their ability to induce cell proliferation of human lymphocytes (Fig. lj. The lectin from Canavalia ensifimis seeds (Con A) was used as a reference lectin (Fig. 2). The doses of the different Lathyrus lectins yielding maximum incorporation of tritiated thymidine are summarized in Table I. The optimal mitogenic doses obtained with the various lectins differed by a factor of 10-30. This range was approximately the same despite the culturing conditions, even though the amplitude of the recorded response was 5-10 times lower when the stimulation was performed under serumfree conditions. From studies of what concentration of monosaccharides was necessary to inhibit the lectin-induced hemagglutination of human red cells (Table II) it was not possible to explain the differences in mitogenic properties that we obtained with the Lathyrus lectins. There was no direct correlation between the requirement of each lectin for a minimum concentration of saccharides giving inhibition of hemagglutination and the ability of each lectin to induce lymphocyte transformation. This might, however, be explained if the membranebound carbohydrate receptors recognized by the Lathyrus lectins on lymphocytes were of the more complexed oligosaccharide types. Accordingly, it was also recently shown that L. ochrus, for example, exhibited the ability to recognize well-defined saccharide sequences on biantennary Nacetyllactosaminic-type glycans (7). In this respect, the limited differences outlined among the primary structures of the Lathyrus lectin subunits (4-6) could sufficiently alter their conformation in such a way that their carbohydrate-binding sites could be slightly modified, thus preventing them from recognizing the membrane-bound receptors to the same extent. The three-domain conformational model recently presented by Olsen (11) in order to explain the

32

BORREBAECK

AND

ROUGti

CONCENTRATION

(pg/ml)

CONCENTRATION

@g/ml)

CC+dCENTRATlON

Qg/ml)

CONCENTRATION

$g/ml)

xK)D xc?0

-3 d h I -m

g tooSC"0.1 CONCENTRATION

+g/mll

-5 0.3

10

3.0

CONCENTRATICN

10

3050

loo

+g/ml)

FIG. 1. The dose-response relation of ten different Luthyrus lectins in RPM1 1640 medium containing 10% fetal calf serum (m) or 0.2% methyl cellulose (0). (A) L aphaca; (B) L. atiiculatus; (C) L ticera; (D) L. him&us; (E) L ochrus (whole lectin); (F) L. OC?WZL.S I (1st isolectin); (G) L ochrus II (2nd isolectin); (H) L. odorutus;(I) L tin&anus; (J) L vernus.

circularly permuted sequence homology that relates Con A to other single- or twochain legume lectins (12), is quite compatible with such a hypothesis. The difference in mitogenic properties of L. ochrus (whole lectin) and the isolated isolectins L. ochrus I and II was, further-

more, interesting to note. The whole lectin had a considerably (3-10 times) better ability to induce cell growth than the individual isolectins had. It seemed that the mixture of the two isolectins, which is represented by the whole lectin, had an synergistic effect on the lymphoid cells. This

MITOGENIC

0.1

0.3

1.0

34

CONCENTRATKJN

CONCENTRATION

10

3050

PROPERTIES

OF Lathyrus

loo

0.l

33

LECTINS

0.3

1.0

3.0

x)

CC+dCENTRATlC+d

(,@,,I)

CONCENTRATION

(pg/ml)

3050

loo

(,,g/ml)

+gjrn~)

FIG. l-Continued

effect was lost upon separation of the isolectins. The difference in mitogenic properties had no obvious explanation unless it was due to differences in the state of molecular aggregation of the lectin preparations. The two isolectins exhibited, fur-

CONCENTRATION

&,/ml,

FIG. 2. The dose-response relation dium as described in Fig. 1.

of Con A in me-

TABLE

I

SUMMARY OF MITOGENIC DOSE YIELDING MAXIMUM CELLULAR RESPONSE Lectin concentration (&ml) Lectin source

RlO

RO

L. aphaca L. articulate L cicera L. ochrus (whole lectin) L ochms I (1st isolectin) L. ochrus II (2nd isolectin) L. odoratus L. hirsutus L. tingitanus L. ?‘wrnus Con A

30 10 >lOO 3 10 30 1100 50 50 50 3

10 3 30 3-10 10 10 10 30 3-10 10-30 1

Note. Measured in medium containing 10% serum (RlO) or under serum-free conditions (RO).

34

BORREBAECK TABLE

MINIMUM

CONCENTRATION

(mM)

OF SUGAR GIVING

sugar

L. aph.

L. art.

L

Mao GlC FI?lC GlcNHz aMet-Mao aMet-Glc GlcNAc

6.25 25 50 100 3.12 12.5 25 25

6.25 25 50 50 3.12 12.5 25 12.5

6.25 25 25 50 3.12 12.5 25 12.5

SlXr0S.e

tic

AND

ROUGfi

II

COMPLETE

INHIBITION

OF HEMAGGLUTINATION

L hir.

L och (w)

L och (I)

L och (II)

L cd.

L

3.12 6.25 12.5 25 0.78 3.12 6.25 6.25

6.25 12.5 25 100 3.12 12.5 25 12.5

6.25 12.5 25 100 3.12 12.5 25 12.5

6.25 12.5 25 100 3.12 12.5 25 12.5

3.12 6.25 12.5 25 0.78 3.12 12.5 12.5

6.25 12.5 25 50 1.56 12.5 12.5 12.5

tin

BY LECTINS

L ver.

Con A

3.12 6.25 12.5 25 0.78 6.25 6.25 6.25

3.12 12.5 6.25 25 0.39 1.56 12.5 12.5

Note.The isolectin and lectin concentrations used were 37.5 fig/ml, these concentrations being 4 to 8 times higher than that producing complete hemagglutination in the last twofold dilution. The following sugars were not inhibitory at final concentration of 200 mbl: o-arabinose, L-fucose. D-galactose, N-acetyl-D-galactosamine, a-methyl-D-galactoside, B-methyl-Dgalaetoside, /3-methyl-D-glueoside, L-rhamnose, D-ribose. Abbreviations used for sugars and derivatives: Man = D-mannose, Glc = D-glucose, Fruc = D-fructose, GlcNH, = D-glUCOSamh2, a Met-Man = a-methyl-D-mannoside, aMet-Glc = a-methyl-D-glucoside, GlcNAc = N-acetyl-D-glucosamine. Abbreviations used for lectins and isolectins: L aph. = I.ath?/rus aphaca, L art. = L articdatus, L tic = L cicera, L hir. = L hirsutus, L och (w) = L ochms, whole lectin, L cxh (I) = L ochrus, isolectin I, L och (II) = L oehrus, isolectin II, L od. = L &at%$ L tin. = L tingitanus, L ver. = L zlernz~s, Con A = concanavalin A.

thermore, an identical carbohydrate specificity towards human, bovine, and hen glycoconjugates (7), with ~(1 - 6)-linked fucose as a major determinant. This study clearly demonstrates the complexity behind the structure-function relation of lectin mitogens, since even phylogenetically closely related Lathyrus lectins exhibited a significant differences in ability to induce cellular transformation. This ability of the Lathyrus lectins probably does not, however, reflect a plant physiological function in vivo but is rather an in vitro consequence.

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