interaction of soybean agglutinin with soybean callus cells

Original Papers

Interaction of Soybean Agglutinin with Soybean Callus Cells

") Department of Biochemistry, University of California, Riverside, California 92521 U.S.A. •. ,') Stauffer Chemical Company, Richmond, California 94804 U.S.A.

Received March 30,1981 . Accepted May 23,1981

Summary Depending on the stage of cell growth, small additions of soybean agglutinin to soybean cells

in culture, can either inhibit or stimulate leucine incorporation into protein. Cell age also affects the amount of soybean agglutinin that can bind to the cells as well as the amount of endogenous soybean agglutinin-like material on the cell surface. It is speculated that the age dependent response of exogenous soybean agglutinin may be related to the ratio of unoccupied: occupied soybean agglutinin receptors on the cell surface.

Key words: Glycine max, lectins, soybean agglutinin, cell culture, receptors, development, protein synthesis, mitogenesis.

Lectins are multivalent carbohydrate binding proteins that can be detected and measured by their ability to agglutinate cells. Cell agglutination is a consequence of the formation of numerous lectin bridges between surface oligosaccharide determinants on neighboring cells. By virtue of this characteristic, lectins have been widely used as a tool to analyze properties of the cell surface (Nicolson, 1974). The binding of lectins to the cell surface has been shown to induce a variety of biological events including cell division (Blomgren and Svedmyr, 1971), and cell differentiation (Southworth, 1975). These observations led investigators to inquire as to whether plant lectins might serve as selective agents to stimulate mitosis of plant cells. Particularly interesting in this regard was the demonstration of a mitogenic effect of soybean agglutinin (SBA) on soybean callus cells (Howard et al., 1977). These experiments were unique in that they examined the effect of a lectin on plant cells derived from the same species from which the lectin was isolated. The present report describes experiments which examine some of the features of the interaction of SBA with soybean cells at different times throughout the cell growth cycle.

Materials and Methods

A soybean callus culture was derived from root tissue (Murashige, 1974) and transferred to liquid Murashige and Skoog culture media as previously described (Uchimiya and Murashige,

Z. Pjlanzenphysiol. Bd. 104. S. 97-102. 1981.

98 ELENA DEL CAMPILLO, JOHN HOWARD and LELAND M. SHANNON

1974). SBA was purified from crude seed extracts by affinity chromatography using N-acetylgalactosamine covalently attached to Sepharose beads (Vector Laboratories, Burlingame, Ca). The adsorbed SBA was displaced with PBS containing 100 mM D-galactose. Following exhaustive dialysis against PBS, the SBA was lyophilized and stored at -20°C. The purified SBA showed a single protein band following SDS gel electrophoresis (Webber and Osborn, 1969).

SBA was filter sterilized and added to growth media to yield a final concentration of 1.5 I-Ig/ml. The initial cell density was 40 mg/ml. Cell growth was determined by measuring changes in the packed cell volume following centrifugation for five minutes at 2000 x g. Protein synthesis was estimated by measuring the incorporation of U-HC leucine into the methanolchloroform-water (12: 5 : 3) insoluble fraction (Ferrari and Widholm, 1973). U-HC leucine was added to the culture medium, and after the addition of the cell inoculum, 0.4 ml of the cell suspension was dispensed into individual culture tubes. At 5 hr intervals the cells were collected onto filter pads, washed three times with methanol-chloroform-water, and the insoluble fraction counted. Quadruplicate assays were run at each time interval.

Rabbit antisera to purified SBA was prepared by methods described previously (Howard et aI., 1979). Lactoperoxidase (501-11, 1 mg/ml) and 250 I-ICi of 1251 (specific activity: 17 Ci/mg; ICN, Irvine, CA) were added to 2 ml of antiSBA-IgG (5 mg/ml). The reaction was initiated by the addition of 25 1-11 of 0.1 M H 20 2. After 30 and 60 min an additional 25 1-11 aliquot of H 20 2 was added. After 90 min the mixture was applied to a Bio-Gel P-100 column (2.5 x 27 cm) and the effluent fractions monitored for IgG using Ouchterlony double diffusion assays. The fractions containing IgG were combined and 1251 measured in a gamma counter.

A suspension of callus cells was centrifuged in a graduated tube at 2000 x g for five minutes. The packed cells were then suspended in phosphate buffered saline (PBS) to obtain a 0.25 % (v/v) suspension of cells. The cell suspension (100 1-11) was then added to a microfuge tube containing one milligram of bovine serum albumin, included to reduce non-specific binding. Cells were then incubated with SBA (100 I-Ig/ml) or PBS for 10 minutes and washed three times in PBS at room temperature. 125I-IgG (100 1-11, 5 mgl ml, specific activity, 5000 cpml mg) was added and incubated with the cells for 30 minutes. The cells were pelleted in a microfuge, washed three times with PBS, and the bound radioactivity was measured using a gamma counter.

Results

Previous studies showed that the addition of 1.5 ~g/ ml of SBA to soybean suspension cultures caused an increase in cell number, mitotic index and rate of 3Hthymidine incorporation into nucleic acid (Howard et aI., 1977). In the present study we examined the effect of SBA on the incorporation of He leucine into proteins. The results of a time course of u-He leucine incorporation into proteins in the absence and presence of 1.5 ~g/ ml SBA are shown in fig. 1. These data reveal that SBA caused a 2 fold increase in the rate of He leucine incorporation into protein. The cell inoculum used in this experiment was harvested during the logarithmic phase of growth.

We next examined the effect of SBA on protein synthesis at different stages of the growth curve. A typical growth curve of soybean cells in suspension culture is seen in fig. 2 A, in which packed cell volume is used as an indicator of growth. For each group of cells harvested at different stages on the growth curve, a time course of UHe leucine incorporation was measured in the absence (control) and in the presence of 1.5 ~g/ ml SBA. The results are depicted in fig. 2 B as percentage of control. There


Interaction of soybean agglutinin with callus cells 99

9 SBA

N

0

6 ~ a. ()

3

6 12 18 24 30 INCUBATION TIME (hrs)

Fig. 1: Effect of SBA on protein synthesis. Soybean cells were harvested during logarithmic growth and transferred to fresh media containing U-HC leucine II! Ci/ml in the absence (0---0) and in the presence of 1.5 I!gl ml SBA (e---e). HC leucine incorporation into proteins was measured at 6 hr intervals. The error bars represent the range of values among the 4 replicates of each assay.

A B 0 E 8

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6 6 0 > o~ 0 -' 100 z -' .... ~ 4 0\

0 " 0 0

0 / r UJ

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5 10 15 20 5 10 15 20 DAYS IN CULTURE

Fig. 2: (A). Growth curve for soybean cells. Suspension cultures of soybean cells were initiated by transferring 1 g of soybean callus tissue to 25 ml Murashige and Skoog media in 125 ml Erlenmeyer flasks. The flasks were continuously shaken at 100-150 rpm on a rotatory shaker at room temperature. At the indicated times the entire contents of the flask were centrifuged for five minutes at 2000 x g and the volume of the packed cells was determined. - (B). Effect of SBA on protein synthesis at different cell ages. Soybean cells were transferred at different stages of the growth cycle to fresh media containing U-HC leucine in the absence (control) and presence of 1.5 I!gl ml SBA. Data are shown as percent of control:

rate of I·C leu incorporation with SBA x 100 = % Control rate of HC leu incorporation without SBA

was no effect of exogenous SBA during the lag phase of growth. Addition of SBA during the initial phase of log growth (day 7) caused an inhibition of the rate of amino acid incorporation over the control. When cells were in the middle of the logarithmic growth phase (day 10), addition of SBA caused a two fold increase in the rate of 14C



leucine incorporation. Finally, addition of SBA to cells approaching the stationary phase did not influence protein synthesis.

The next experiment was undertaken to determine the amount of exogenous SBA capable of binding to cultured soybean cells harvested at various times during the growth cycle. In these studies iodinated (1251) IgG against SBA was added to soybean callus cells which had been previously incubated with or wihtout SBA. The amount of 1251_lgG bound to cells which had been incubated in the absence of exogenous SBA (control), represents a measure of endogenous SBA-like material on the cell surface. The increase in 1251-lgG bound to cells treated with SBA represents a measure of unoccupied SBA receptor sites. When 1251-labeled pre-immune IgG was substituted for 1251-SBA IgG, negligible radioactivity was bound. Maximal endogenous SBA like material was present on cells collected in the log phase of growth. As cells approached stationary growth phase, the SBA content of the media increased dramatically (data

200r---------------------------~

180

160

140

120

o ~IOO o m

b 80 o m >= z <X 60

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40

20

3 5 7 9 ~ 17 DAYS IN CULTURE

Fig. 3: Effect of culture age on the amount of SBA Ab bound to cells presviously incubated with (0-----0) or without (e-----e) SBA. Values shown represent the average of 5 replicates; deviation among replicates was less than 5 %.

not shown), presumably a consequence of the release of endogenous SBA from the cell surface. The amount of exogenous SBA which bound to the cell was also highest during log phase of growth. Cells in lag phase bound only a small amount and cells in stationary phase were totally unable to bind exogenous SBA.


Interaction of soybean agglutinin with callus cells 101

Discussion

The mitogenic effect of SBA on soybean cells growing in suspension culture was followed by measuring the incorporation of U-HC leucine into protein. The rationale for this approach was based upon the premise that whatever the molecular mechanism of mitogenesis, it should include in the sequence of events, the stimulation of protein synthesis. There was a two fold increase in the rate of 14C leucine incorporation into proteins in SBA treated cells compared with control cells. The mitogenic effect of SBA on soybean callus cells appears to be highly dependent on the age of the cell. Addition of SBA to cells at the beginning of log phase caused an inhibition in the rate of amino acid incorporation into proteins. Addition of SBA to cells in the middle of the logarithmic phase caused a two fold increase in the rate of amino acid incorporation into proteins. No effect of SBA was observed when the cells were in the stationary phase of the growth cycle. These experiments clearly show that, depending on the age of the cell, SBA can inhibit, stimulate, or not affect the rate of 14C leucine incorporation into proteins.

In our previous paper (Howard et aI., 1977), SBA was shown to increase cell number, cell weight, mitotic index and DNA synthesis. An increase in protein synthesis is clearly involved in these responses. In this paper SBA was shown to increase HC leucine inorporation into protein. This latter effect may have resulted from an increase in HC leucine transport and/or an increase in protein synthesis.

Cell age also influenced the amount of endogenous SBA-like material on the cell surface as well as the amount of exogenous SBA that could bind to the cells. We believe the latter response reflects the quantity of unoccupied SBA receptors on the cell surface. The developmental effect on apparent unoccupied SBA receptors could reflect a change in their number or a change in their affinity and/or availability to SBA. These observations are consistent with earlier developmental studies on sea urchin embryos and neural retina cells in which it was shown that the number of lectin binding sites changed during normal development (Krach et aI., 1974 and Kleinschuster and Moscona, 1972).

The biological effects of SBA on soybean cells could be explained if we assume that an optimal ratio of unoccupied receptors to occupied receptors must be maintained in order to observe the mitogenic response. This hypothesis was proposed to explain the mitogenic effect of Con A on lymphocytes (Anderson et aI., 1972; Betal and Van Den Berg, 1971; Ibar and Sachs, 1973). In the ConA-lymphocyte system only 5 % of the available lectin sites need be occupied to induce mitosis. In fact, a massive saturation of the receptors caused an inhibition of the normal cell function.

During the beginning of the exponential phase of growth, a large population of cells start to actively divide. At this point in the cell cycle the exogenous SBA is su"fficient to cause a massive saturation of the unoccupied SBA surface receptors and inhibit normal cell function. When the cells are in the middle of the exponential phase, new cells with large numbers of unoccupied receptor sites appear such that



even with addition of exogenous SBA, an optimal ratio of unoccupied to occupied receptors is maintained. Cells in stationary growth phase appear non-responsive to exogenous SBA, presumably because they totally lack unoccupied SBA receptors.

It is important to know whether the response observed with SBA has any physiological significance. The soybean cells contain the genetic information needed for the synthesis of endogenous SBA, thus SBA is not a foreign protein to these cells. Also, the quantity of SBA that imparts the maximal mitogenic response is relatively low (1.5 !lg/ml), which supports the idea that the effects noted with exogenous SBA could also occur with endogenous SBA. There is, however, the possibility that these observations are irrelevant to what occurs in vivo. Exogenous SBA may be acting merely as a membrane perturbing agent, as it does in mammalian cells, and the responses observed, while real, are not related to the physiological function of lectins. Nevertheless, the SBA-induced responses of soybean cells provide a powerful research tool for studying plant cell surfaces and their variability with development.

This work was supported in part by the National Science Foundation (PCM 79-01450-01).

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interaction of soybean agglutinin with soybean callus cells

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