THE LECTINS AND LECTIN-LIKE PROTEINS OF TEPARY BEANS (PHASEOLUS ACUTIFOLIUS) AND TEPARY-

COMMON BEAN (PHASEOLUS VULGARIS) HYBRIDS

ELVIRA G. DE MEJIA'.', CHARLES N. HANKINS, OCTAVIO PAREDES-LOPEZ', and LELAND M. SHANNON'

Department of Biochemistry University of California at Riverside

Riverside, California 92521

Received for Publication June 28, 1989 Accepted for Publication October 5, 1989

ABSTRACT

High levels of lectin activity were found in sixty cultivated and ten wild tepary (Phaseolus acutifolius) accessions. No lectin deficient varieties were observed and all examples studied contained both the phytohemagglutinin-E and L-like lectins previously described (Pusztai et al. 1987). There appeared to be no obvious differences between the wild and cultivated forms of the tepary lectins and all teparies studied contained lectin-like proteins in addition to the tepary lectins. One of the lectin related proteins (40 Kdalton subunit) was present in all teparies and may be comparable to arcelin, a lectin found in certain wild accessions of Phaselus vulgaris. All wild teparies contained a lectin related polypeptide of about 34 Kdaltons which appears to distinguish the wild teparies from the cultivated forms. Three tepary-common bean hybrids were examined and one hybrid was found to be expressing both tepary and common bean lectin genes.

INTRODUCTION

The tepary bean (Phaseolus acutifolius) is an annual legume adapted to the and and semi arid regions of the world, particularly North America, where it has been cultivated for many centuries (Nabhan and Felger 1978). Teparies thrive in high temperatures, are very drought resistant, and possess resistance to mi- crobial predation (Thomas et al. 1983; Coyne and Schuster 1973). Also, teparies

'Centro de Investigacion en Alimentacion y Desarrollo Hermosillo, Sonora, Mexico *Centre de Investigaciones y Estudios Avanzados-IPN Irapuato, Guanajuato, Mexico 'Address all correspondence to: Dr. Leland M. Shannon, Department of Biochemistry, University of California, Riverside, Riverside, California 92521-0129.

Journal of Food Biochemistry 14 (1990) 117-126. A// Rights Reserved. G Copyright 1990 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 1 I7

118 ELVIRA G . de MEJIA. et. al.

appear to be resistant to certain beetles which thrive on common beans stored in the same location (unpublished observation).

Interest in these close relatives of the common bean (Phaseolus vulgaris) centers on both their value as an arid land crop and as a source of stress resistant germ plasm. Tepary-common bean hybrids have been produced (Pratt 1983) and it appears that the stable introduction of tepary traits into common beans (or vice versa) by hybridization is feasible (Thomas and Waines 1980, 1984).

Like common beans, teparies are quite toxic to man and animals (Gonzalez- Garza et a/ . 1982; Thorn et al. 1983) in the raw form, presumably due to the PHA'-like lectins in teparies (Pusztai et a/ . 1987). Since lectins (phytohemag- glutinins) may contribute desirable traits (large quantity of reserve protein and confer pest resistance) and undesirable traits (toxicity), they are of interest nutritionally as well as agronomically. Lectins have been purified from P . acu- tifolius L . and shown to be extremely similar, both structurally and immuno- logically, to PHA from P . vulgaris (Pusztai et al. 1987). The teparies, like common beans, contain an erythroagglutinin (PAL-E) and a lymphoagglutinin (PAL-L). Little information is available on the lectin content or distribution in the many wild and cultivated tepary varieties available and nothing is known about the lectins of tepary-common bean hybrids. Also, it is not known if teparies contain a protein comparable to arcelin (Osborn et al. 1988), the PHA-related lectin that confers high resistance to certain beetles in some wild bean accessions (Osborn et al. 1986).

In this report, we survey the lectins and lectin-like materials (CRM) in seventy teparies including ten wild accessions. We also examine these proteins in three different F, backcross hybrids of P . acutifolius and P. vulgaris.

MATERIALS AND METHODS

Seeds

Tepary bean, common bean, and hybrid bean seeds were from the legume collection at University of California, Riverside, CA. All were cultivated at UCR in the Summer of 1987 and kindly supplied to us by Dr. Giles Waines, De- partment of Botany and Plant Sciences, UCR. The cultivated tepary accessions studied were PI-319-443 (Blanco), PI-146800, L-242-6, L-578, L-242-12, L-242-10, L-242-11, L-246-14, L-242-45, L-179, L-242-7, L-395, L-242-16, L-371, L-373, L-246-19, L-242,38, L-242-36, L-242-40, L-242-25, L-242-29, PI-239056, PI-321637, L-246-12, L-242-46, PI-310-606, L-242-22, L-246-9, L-242-43, L-242-4, L-278, L-242-24, L-172, PI-321-637-1, PI-310-801, PI-321631, L-246-10, L-246-3, L-171, L-246-22B, L-246-23, L-246-1, L-173, L-170, L-392, L-246-5, L-169, L-376, L-246, PI-301801, G40035, PI-321638, (340036, L-242-26, PI-10606, G40034 and PI-313814. The wild teparies were:

LECTINS OF TEPARY BEANS 119

L-124, PI-319441, L-137, L-177, PI-19439, PI-319444, PI-319438, PI-319442, PI-3 19440 and PI-263590.

Extractions

Dry seeds were ground into flour with a Wiley mill or by hand in a mortar and pestle. For active lectin, flour was extracted overnight at 5°C with PBS (10 mWg flour) and then centrifuged at 12,000 X g for 20 min to yield the crude extract. Crude extracts were dialyzed overnight against PBS and stored at - 20°C.

For total seed protein extractions, flour was boiled with SDS PAGE sample buffer (10 mLig flour) for 5 min and centrifuged as above, to give a crude SDS extract. In the cases of hybrids, single seeds were extracted.

Hemagglutinin Assays

Lectins were assayed by recording the number of serial two-fold dilutions of the extract (in PBS) required to eliminate the ability to agglutinate a 2% sus- pension of washed rabbit or human erythrocytes.

Electrophoresis

Vertical 12% slab SDS-PAGE was carried out on 8 X 10 cm gels (Hoefer) using the method of Laemmli (Laemmli 1970). Electroblotting onto nitrocellulose was done with a Hoefer transphor apparatus according to the manufacturer's directions. Gels were stained with either Coomassie Brilliant Blue R-250 (Fair- banks et al. 1971) or periodic acid-Schiff's (Zacharius et al. 1969). Western blots were stained with rabbit anti sera against PHA and then goat antirabbit IgG-alkaline phosphatase (Sigma). The procedure was that described by Knecht and Diamond (Knecht and Diamond 1984) except an overnight blocking step was added using 1.4 g/mL nonfat dry milk (Carnation) in water. Sodium azide (0.1%) was used as a preservative in blocking solutions.

Chromatography

For lectin purification, crude extracts were chromatographed on Fetuin-agarose (Sigma) as described by Pusztai et al. (1987). Gel filtration was done on Se- phacryl S-200 (Pharmacia).

RESULTS AND DISCUSSION

Seventy teparies were examined including sixty cultivated accessions and ten wild accessions. Additionally, we examined several common bean varieties in- cluding Ica pijao (black bean) and NB585 (haricot), which were the Phaseolus

120 ELVIRA G. de MEJIA, et. a1

vulgaris parents of the three hybrids studied. The tepary parent (PI 319-443) of the hybrids was a cultivated variety with small white seeds.

The hemagglutinin titers of all the teparies were very high (2 12 serial two- fold dilutions) using washed rabbit erythrocytes and all were active with human A, B or 0 type blood cells. None of the teparies was observed to have an unusual hemagglutinin activity with respect to either titer or specificity. No lectin deficient varieties, as are known with common beans, were found in this sampling. Sam- ples of both wild and cultivated beans were found to give identical fetuin-agarose chromatograms, hence both wild and cultivated teparies contain PAL-E and PAL-L. The lectins purified from wild teparies were indistinguishable from those of cultivated varieties by SDS-PAGE.

Initially, we screened crude SDS extracts of all beans by SDS-PAGE. Gels (or electroblots of gels) were stained with Coomassie Brilliant Blue R-250 to reveal total polypeptides, periodic acid-Schiff's (P.A.S.) to show the glycopep- tides, and immunologically to identify lectin subunits and lectin related (CRM) polypeptides. These studies were done with extracts of dry flour made with boiling SDS buffers. This method insured that we would not miss any soluble proteins and obtain a more nearly complete visualization of all the polypeptides in seeds. Preliminary Western blots using anti PHA-E and anti PHA-L sera indicated that both sera recognized the same set of polypeptides in tepary extracts as well as in several common beans examined. Western blots of common bean crude extracts revealed that the sera reacted with the known PHA subunits in the 33-40 Kda range, depending on variety, and also reacted weakly with a 45 Kda polypeptide in some varieties. PHA subunit banding patterns are known to vary considerably from variety to variety, and other lectin-like proteins have been found in certain beans (Osborn et al. 1986; Pusztai et al. 1981; Brown et al. 1982; Hoffman 1984). We chose to screen with anti PHA-E sera because a good supply of high titer sera was available and it appeared to cross react about equally well with purified PAL-E and PAL-L.

Overall, the tepary seed polypeptide patterns were, as expected, extremely similar, especially among the cultivated types. Of 60 cultivated teparies exam- ined, at least 50 appeared to have essentially identical patterns, like the three representative examples shown in Fig. 1. These teparies contain four major lectin CRMs with the approximate sizes as shown in Fig. 1C. CRM band 1 is the weakest and is sometimes absent; CRM bands 1 and 2 are not exactly coincident with any major Coomassie or P.A.S. staining polypeptides, although band 2 is difficult to distinguish from the major Coomassie staining polypeptide at about 45 Kda. CRM bands 3 and 4 appear to be major glycoproteins while band 5 is weakly P.A.S. positive. The other cultivated samples examined showed CRM patterns which were almost the same (and in some cases may only appear slightly different due to experimental variability). Some of these other types are shown in Fig. 2. Note that these teparies all have the same CRM bands as those in

LECTINS OF TEPARY BEANS 121

A B c

--I ( 5 4 K d a )

-3 ( 4 0 K d a ) -4 (3 I Kda) 1 5 ( 2 9 K d a f

-2 ( 4 5 K d O )

FIG. 1 . SDS-PAGE OF THREE TYPICAL CULTIVATED TEPARIES (1,2,3) A Periodic acid-Schiff's base stained; B. Coomassie Brilliant Blue R-250 stained,

C. Irnrnunostained (Western).

Fig. 1C but they differ either by having an additional weak CRM band at about 34 Kda as seen with numbers 3, 4 and 5 or (and) the relative strength of certain bands appear noticeably different. For example, CRM band 1 (54 Kda) is quite variable in both size and intensity compared to the types shown in Fig. 1. Band 2 may also be somewhat variable. The 40 Kda CRM band appears to be the least variable band both in molecular weight and staining intensity in all the cultivated samples.

Overall, wild teparies contain the same CRM bands as the cultivated types. As seen in Fig. 3, however, there is considerably more variation in the intensity

I 2 3 4 5 6

FIG. 2. WESTERN BLOT OF SDS GEL OF SIX DIFFERENT CULTIVATED TEPARY EXTRACTS

From 1 to 6, they are: L-242-24, (3-40035, L-242-40, L-246-19, PI-321631 and PI-146800.

122 ELVIRA G. de MEJIA, et. a1

I 2 3 4 5 6 7 8 9 1 0

FIG. 3. WESTERN BLOT OF SDS GEL OF TEN DIFFERENT WILD TEPARY EXTRACTS From 1 to 10, they are: G-40016, L-177, PI-263590, PI-319442, PI-319444, L-137, PI-319440, L-124, PI-319438 and PI-319439.

of staining of most of the bands and all of the wild teparies contained a major CRM band at about 33-36 Kda which appears to be characteristic of the wild forms. Whether or not this band is the same as the weak CRM band 6 seen in cultivated forms will require further study. Note that, as was also the case with the cultivated types, the 40 Kda band is virtually invariant in size and intensity. All of the other CRM bands show at least some degree of variation among the wild teparies. For example, the 45 Kda band 2 appears as a doublet (difficult to see in photograph) in samples 5 and 8, while in sample 6 this band is an abnormally strong CRM. The high variability of CRM band 2, both in molecular weight and intensity of staining, set it apart from the major storage protein subunits which appear essentially invariant by protein staining in all teparies. CRM bands 4 and 5 also vary considerably and, in samples 3 and 5 , band 5 of this doublet is very weak or possibly not present at all.

In an effort to identify the active PAL subunits and other possible lectins, we examined tepary extracts by affinity chromatography on fetuin-Sepharose. The chromatographic patterns (data not shown) revealed that both wild and cultivated tepary extracts contain the weakly interacting PAL-L and strongly interacting PAL-E forms described by Pusztai et al. (1987). The wild and cultivated forms of each of these lectins appeared to be the same, at least by SDS-PAGE, and in neither wild nor cultivated forms were the E and L types of subunits separable by SDS-PAGE. Western blots of the affinity column fractions showed that band 4 (about 31 Kda) represents both of the tepary lectin subunits and none of the other CRM bands interact with this matrix.

In an effort to further characterize the nature of the various CRM polypeptides we examined their behavior in an undenatured state by gel filtration. Western blots of fractions from across a calibrated Sephacryl-S-200 column allowed us to obtain native molecular weights for all the CRM bands. As expected, the

LECTINS OF TEPARY BEANS 123

tetrameric PAL lectins ran at about 115-120 Kda. The 53 Kda CRM (band 1) ran as a native protein of about 35 Kda , which indicates that this CRM either interacts with the column matrix or runs abnormally slow on SDS gels. The 45 Kda CRM (band 2 ) had a native molecular weight of about 180 Kda; thus, if this protein is not associated with polypeptides which are not CRMs, then it is a tetrameric protein like the lectins. CRM band 3 (40 Kda) ran at about YO Kda on gel filtration suggesting a dimeric structure. Since this CRM has a subunit size and apparent dimeric structure similar to arcelin from common beans, it is possible that this CRM may be a comparable protein from tepary. Although band 3 migrated almost identically to one of the subunits of PHA (Fig. 4), it is not a subunit of PAL nor does it interact with the affinity column used in these studies. Whether the 40 Kda CRM is responsible for part (or all) of the observed beetle resistance of teparies must await further study. CRM band 5 , which was present in almost all the teparies studied, gave a broad smear on gel filtration, indicative of multiple aggregation states. Thus, band 5, although very similar in size and immunoreactivity to the PAL subunits, is not a PAL subunit nor does it form stable dimeric or tetrameric structures.

The anti PHA sera used in these studies could conceivably contain antibodies against carbohydrate determinants and we have not ruled out this possibility. However, there are many glycoproteins in both P . vulgaris and tepary extracts which are not recognized by this sera and all CRM bands seen are not glyco- proteins. Also, based upon the recent observations by Lauriere et al. (1Y89), P . vulgaris extracts contain many glycopeptides containing PHA-like carbohy-

I T B H C B

rl[ m: T B H CB T B H CB

FIG. 4. WESTERN BLOTS OF SDS GELS OF THREE TEPARY-COMMON BEAN HYBRID CROSSES

TB: Teparty bean parent. H: Hybrid. CB: common bean parent

Tepary blanco (PI-319-443) x Ica pijao (black bean) Tepary blanco (Pi-319.443) x Icu pijuo (black bean)

Tepary blanco (PI-319-443) X Haricot (NBSSS)

I: 11:

111:

124 ELVIRA G. de MEJIA, et. al.

drate side chains. We do not see anything resembling the complex Western blots observed by these authors with our anti sera.

Fortunately, in addition to the many teparies available, seeds from three sep- arate hybrid crosses were also available. We examined the tepary parents, com- mon bean parents and the hybrids together for clarity. The results of SDS PAGE and Western blotting of crude extracts prepared from individual parent and hybrid seeds is given in Fig. 4. The P . vulgaris parents appear identical to one another and show a doublet CRM in the region of band 2 as well as the PHA subunits at about 33-35 Kda (band 6) and 40 Kda (band 3 region). A detailed examination of the polypeptide patterns reveal that the hybrids are distinct from either parent in all three cases; however, the major seed proteins of hybrids I and I1 appear almost identical to their P . vulgaris parent. Hybrid 111, on the other hand, appears to have a more complicated pattern which contains major polypeptides from both parents. Hybrids I and I1 do not appear to contain any tepary lectin subunits but are expressing PHA genes while hybrid 111 is clearly expressing both tepary lectin genes and PHA genes. From the Western blots of these gels, it is also clear that the CRM pattern of hybrid I11 is a mixture of those of both parents. Thus, this hybrid is expressing lectin CRM genes of both parents in addition to the lectin genes.

Based upon these studies, it appears that lectin and lectin-like polypeptides are ubiquitous and can be easily detected in teparies, and their hybrids by Western blotting of extracts from individual seeds (or parts of seeds). Thus, experiments designed to assess possible correlations between them and animal toxicity or specific stress resistance are feasible. Since PHA is partially responsible for the toxicity of common beans, PAL is likely to be the major protein toxin of tepary seeds. The fact that teparies contain several additional lectin-like proteins sug- gests that they may contain several types of toxins, perhaps with specificities for insects and microbes as well as mammals. Although a lectin negative variety of tepary was not found, such varieties are available in common beans and may be found in teparies with additional screening or produced from hybrid crosses.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Giles Waines for providing bean seeds for this study and Dr. Claire Thomas for the valuable hybrid seeds. We also wish to acknowledge the support of the U.S. Agency for International Development and the University of California UC-Mexus Program.

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