Inhibition of acyltransferase in lymphocytes by concanavalin A

305

Blochtmwa et Bmphystca Acta, 6 2 9 ( 1 9 8 0 ) 3 0 5 - - 3 1 6 © E l s e v m r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 29244

INHIBITION OF A C Y L T R A N S F E R A S E IN LYMPHOCYTES BY CONCANAVALIN A

PAUL DOBSON * and ALAN MELLORS

Guelph-Waterloo Centre for Graduate Work zn Chemistry, Department of Chemistry, Unzverszty of Guelph, Guelph, Ontarzo NIG 2W1 (Canada) ( R e c e i v e d July 2 0 t h , 1 9 7 9 ) (Revised m a n u s c r i p t received D e c e m b e r 1 7 t h , 1 9 7 9 )

Key words Concanavahn A, Acyltransferase znh~b~tzon, (Lymphocyte)

Summary The effects of concanavalin A and succinylated concanavalin A on the transformation of mouse splenic lymphocytes, and on early biochemical events in the transformation, were compared. 1. The transformation of lymphocytes is biphasic with respect to concanavalin A concentration with optimal activation at a b o u t 1 ~zg/ml. Activation b y succinyl concanavalin A is not biphasic over a range of lectin concentration of 1--16 ~zg/ml. 2. In intact lymphocytes cultured for 4 h, the enzyme Acyl-CoA:l-acylglycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) was n o t activated b y concanavalin A b u t was inhibited at all concentrations tested, and was a b o u t 60% inhibited at 16 pg concanavalin A per ml. Succinyl concanavalin A gave little or no inhibition at similar concentrations. 3. L y m p h o c y t e s become committed to divide while their acyltransferase activities are markedly inhibited b y concanavalin A. 4. The inhibition of acyltransferase by concanavalin A can be lifted by displacing the lectln from the cells by ~-methylmannoside. Lowered enzyme activity is n o t caused by cell agglutination or by direct cross-linking of lectin receptors. It is unlikely that the inhibition of acyltransferase is due to indirect cross-linkang via the cytoskeleton since colchicine did not reverse the inhibition. 5. The inhibition of acyltransferase and the reduced stimulation of transformation by higher levels of concanavalin A appear to be due to hydrophobic interaction of the lectin with the plasma membrane, as shown b y liposome aggregation studies. * Present address" Dlwslon o f Pharmacology, I.C.I. Americas Inc., Wilmington, DE 19897, U S A Abbreviation, Hepes, 4-(2-hydroxyethyl)-l-plperazmeethanesulfomc acid.

306 Introduction Mltogenic lectins frequently show blphasm effects, as a functmn of their concentration, on the transformation of T-lymphocytes. For example, concanavalin A exhibits an optimal concentratmn for mltogenesss of T-lymphocytes, whmh for mouse spleen cells is a b o u t 1 pg/ml. Above this concentration of lectin there is decreased shmulation of mltogenesis, increased toxicxty and mhlbitmn of the mlgratmn of lectin receptors (capping) [ 1]. Succinylated concanavalin A, a divalent dimer, is mitogemc without showing a biphasic concentratmn effect and is much less toxic at higher mltogen concentratmns [2]. We have investigated the relative effects of concanavahn A and succinyl concanavalin A on some early events m l y m p h o c y t e s undergoing transformation. In particular we have studied the effects of these lectins on the membrane enzyme Acyl-CoA:l-acylglycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) which Ferber and Resch have imphcated in mitogenesls [3]. We have examined the effect of these lectins on acyltransferase activity and conclude that in mtact mouse l y m p h o c y t e s there is n o t activation but a marked inhibition of acyltransferase by concanavalin A, due to interactmn of h y d r o p h o b m regmns of the lectin with the plasma membrane. We fred that l y m p h o c y t e s become committed to diwde while their acyltransferase levels are markedly inhibited b y concanavahn A. Materials and Methods

(1) Chemicals Succinyl concanavalin A was obtained from Polyscmnces (Warrington, PA); other lectms and biochemicals from Sigma Chemical Co. (St. Louis, MO); media from Gibco (Grand Island, NY); anti-concanavalin A serum containing 1.9 mg antibody per ml serum from Miles Laboratorms (Elkhart, IN); isotopes and radio-assay materials for cyclic AMP determinations from Amersham Corp., (Oakwlle, Ontario) Canada; phosphodiesterase from Boehringer-Mannheim Corp. (Ville-St. Laurent, Quebec) Canada. All lectms were chromatographically pure as determined b y polyacrylamide gel electrophoresis, except for Phaseolus vulgaris lectin whmh contained two major bands and two minor bands. Lummolchicine was prepared by exposing colchmme m 95% ethanol to ultraviolet light in a Beckman DB-GT s p e c t r o p h o t o m e t e r according to the m e t h o d of Wilson and Frmdkin [4]. (2) Substrates. [32P]phosphatidylcholine was obtained from a rat injected with 10--50 mCi of [32P]phosphoric acid (carrier free) by the m e t h o d described previously [5] and [32P]lysophosphatldylchohne was prepared from [32P]phosphatidylcholine using Crotalus adamanteus venom [6] and was characterized b y phosphate assay [7], scintillation counting and thin layer chromatography. Typical preparations were greater than 98% pure and had an initial specific activity of approx. 1 Ci/mol. (3) Lymphocyte isolation. Spleens were removed from 25 g inbred white mine and gently homogemzed by hand using a Teflon-glass homogenizer, m 20 ml of Hank's balanced salt solution. The homogenate was allowed to settle for 5 mm to remove debris and the supernatant was centrifuged in a clinmal centrifuge at 400 × g for 10 min. The pellet (PI) was cleared of platelets, dead

307 cells and red blood cells by hypotonic lysis [8] or density gradient separation [9], and resuspended in medium RPMI 1640 or Eagle's minimal essential medium. The resulting lymphocyte-rich suspensions were counted in a hemac y t o m e t e r and used immediately. Cell concentration was adjusted to 106--107 m1-1 and cell viability determined b y trypan blue exclusion or b y SlCr release. Only preparations with viabilities greater than 95% were used further. (4) Viability assays by S~Cr release. Cell viability in short-term culture (less than 4 h) was measured by adapting a S~Cr release assay designed for measuring target cell death in cytotoxicity assays [10,11]. Experiments in which cell viability was correlated with acyltransferase inhibition were performed concurrently using aliquots of the same cell preparation. L y m p h o c y t e s (5 • 10 s) were suspended in 30 ml of medium RPMI 1640 containing 5% heat-inactivated fetal calf serum. The cell suspension was incubated with 500 pCi of sterile sodium [S~Cr]chromate (3.4 pg) for 1 h at 37°C. The suspension was centrifuged at 400 × g for 10 min, and the pellet was washed three times with 20 ml of medium RPMI 1640 without fetal calf serum, to remove excess chromium and dead cells. The cells were resuspended in RPMI 1640 medium and incubated with drugs or mitogens in the same manner as in the acyltransferase assay. Incubation was terminated 1--4 h later by centrifugation at 400 × g for 10 min and 200 /A of the supernatant removed for counting in a Nuclear Chicago 4233 Auto Gamma system. Total chromate release was measured in control tubes by freezing and thawing the preparations three times. Spontaneous release was always less than 15% of total release. Viability was calculated by the following equation: percent viable = 1 -- (test release -- spontaneous release) × 100 (freeze-thaw -- spontaneous release)

(5) [3H]Thymidine uptake. L y m p h o c y t e blastogenesis was evaluated b y measuring the uptake of [3H]thymidine into cellular trichloroacetic acidinsoluble material [12]. To 106 lymphocytes in 1 ml of RPMI 1640 medium containing 5 mM sodium bicarbonate, penicillin (100 units/ml), streptomycin (100 ~g/ml) (pH 7.6), was added 50 #1 of Hank's solution containing 0--100 pg of concanavalin A. In pulse stimulation experiments, 0.1 M a-methylmannoside was added to the incubation tubes at various times after the addition of concanavalin A to displace the lectin from the l y m p h o c y t e surface [ 13 ]. Appropriate controls in which a-methylmannoside was added at the same time as concanavalin A and in which [3H]concanavalin A was used to monitor binding to the l y m p h o c y t e surface indicated that 0.1 M a-methylmannoside displaced cell-bound lectin to non-mitogenic levels. The tubes were incubated for 46 h at 37°C under a humidified atmosphere of 5% CO2. Cell viability was checked b y trypan blue exclusion and the tubes were pulsed with 1 #Ci of [3H]thymidine (6.7 Ci/mmol) for 2 h. The cells were harvested on filter papers in a Titertek Cell Harvester (Flow Laboratories). The filter papers were washed with 5% cold trichloroacetic acid, dried and placed in scintillation vials. 5 ml of scintillation fluid (15 g 2,5-diphenyloxazole in 3.8 1 toluene) were added to each vial and the samples counted in a Beckman LS 7000 scintillation counter. (6) Cyclic AMP determination. Cyclic 3',5'-AMP levels in l y m p h o c y t e suspensions were assayed by the protein-binding method of Gillman [14] using a

308 cyclic AMP Assay Kit (Amersham). The precision of this assay was established by using internal standards to monitor recovery efficiency and by treating aliquots with phosphodiesterase (Boehringer-Mannheim) to obtain appropriate blank values [15]. (7) [14C]Acetate incorporation. L y m p h o c y t e lipid metabolism was evaluated by measuring the uptake of precursor [14C]acetate into various lipids [13]. To 107 lymphocytes, in 1 ml of RPMI 1640 medium containing 25 mM Hepes buffer (pH 7.4), was added 50 pl of Hank's solution containing 0--100 pg of concanavalin A and 50 #l of Hank's solution containing 25 nmol of [14C]acetate (58 Ci/mol). The mixture was incubated 4 h at 37°C in a shaking waterbath. The incubation was terminated by the addition of 2 ml of cold 5% trichloroacetic acid and the mixture was centrifuged at 1000 × g for 10 min. The pellet was washed twice with cold 5% trichloroacetic acid, and subjected to lipid extraction and thin layer chromatography. (8) Acyltransferase assay. Acyltransferase was assayed by measuring the incorporation of oleoyl-CoA into lyso[32P]phosphatidylcholine to form [32p]_ phosphatidylcholine, as described previously [ 16]. (9) Lipid extraction and separation. Lipids were extracted from l y m p h o c y t e trichloroacetic acid precipitates and separated by thin-layer chromatography [16]. Radioactivity was determined by liquid scintillation counting in a Beckman LS-255 Counter. (10) Liposomes aggregation and lymphocyte agglutination. Unilamellar liposomes were prepared by sonication of dipalmitoyl phosphatidylcholine liposomes, by the m e t h o d of van der Bosch and McConnell [17]. The suspension was made 4 mM with respect to EDTA to stabilize the vesicles, followed by centrifugation at 12 000 × g for 20 min at 4°C to remove insoluble material and multilamellar liposomes [18]. The binding of lectins to lipid vesicles via h y d r o p h o b i c interactions was examined by measuring their ability to increase the turbidity of unilamellar dipalmitoyl phosphatidylcholine vesicle suspensions [18,19]. To 1 ml of the lipid vesicle solution (1.0 mg/ml) was added 1 ml of the lectin (0.2--10.0 mg/ml) in the same buffer, and the mixture was briefly vortexed. The mixture was incubated at 25vC and lectin-induced vesicle aggregation was determined at various times (0--4 h) by measuring increases in turbidity at 450 nm in a Beckman DB-GT spectrophotometer. Lectin-induced l y m p h o c y t e agglutination was assessed by light microscopy of cell populations (5 • 106 m1-1) after incubation at 37°C for 4 h, and scored using a hemacytometer. (11) Protein assays. Protein was assayed by the m e t h o d of Lowry et al. [20] using bovine serum albumin as a standard. Results

Fig. 1. shows the effects of a range of concentrations of concanavalin A and succinyl concanavalin A on the incorporation of [3H]thymidine into mouse splenic lymphocytes. Both lectins induced l y m p h o c y t e activation as measured after 4 8 h culture. Concanavalin A induced a maximal mitogenic effect between 0.5 and 2.0 pg/ml, and above these concentrations progressively less activation was observed. Succinyl concanavalin A exhibited a comparable

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Fig. 1. T h y m i d i n e i n c o r p o r a t i o n i n t o m o u s e s p l e e n l y m p h o c y t e s as a f u n c t i o n o f c o n c a n a v a l i n A a n d succ i n y l a t e d c o n c a n a v a l l n A c o n c e n t r a t i o n s . T h e i n c o r p o r a t i o n of [ 3 H ] t h y m i d i n e i n t o t r i c h l o r o a c e t i c acidinsoluble cellular m a t e r i a l was m e a s u r e d b y a d d i n g [ 3 H ] t h y m i d i n e 46 h a f t e r l e c t i n a d d i t i o n , a n d i n c u b a t ing t h e cells f o r 2 h. T h e solid line is f o r c o n c a n a v a l i n A a d d i t i o n a n d t h e b r o k e n line f o r s u c c i n y l a t e d c o n c a n a v a l i n A a d d i t i o n . T h e p o i n t s s h o w n are t h e m e a n s w i t h s t a n d a r d d e v i a t i o n s f o r f o u r d e t e r m i n a tions. T h y m i d i n e u p t a k e u n i t s arc c p m X 10 - 3 . Fig. 2. Cyclic 3~,5'-AMP levels in m o u s e s p l e e n l y m p h o c y t e s f o l l o w i n g l e c t i n a d m i n i s t r a t i o n . T o t a l c y c l i c 3t,5t-AMP was e s t i m a t e d 10 m i n a f t e r lectin a d m i n i s t r a t i o n a n d is e x p r e s s e d as p m o l p e r 107 cells. T h e solid line is for c o n c a n a v a l l n A a n d t h e b r o k e n line, s u c c i n y l a t e d c o n c a n a v a l i n A . E a c h p o i n t is t h e m e a n with standard deviation for three estimates.

mitogenic action at low concentrations, but did not exhibit decreased activation at higher lectin concentrations (greater than 2 ~g/ml). Lymphocyte viability as measured by trypan blue exclusion indicated that high concanavalin A concentrations were more toxic to the cells (10 pg/ml gave 10% viability at 48 h) than similar concentrations of succinyl concanavalin A (10 pg/ml gave 65% viability at 48 h). Cyclic nucleotides have been implicated in the early events of lymphocyte activation, and cyclic AMP levels in intact lymphocytes, 10 min after exposure to the two lectins, were compared. Concanavalin A produced a concentrationdependent increase in cyclic AMP whereas succinyl concanavalin A produced no significant changes in cyclic AMP levels at 10 min (Fig. 2), or within the first 2 h of incubation. Early lipid metabolism changes in activated lymphocytes were examined by monitoring the uptake of [i4C]acetate into lymphocyte lipids (Fig. 3). Increased uptake into all lipid fractions was induced by optimal mitogen concentrations of concanavalin A within 3--6 h of the addition of the lectin. Triacylglycerol, phosphatidylethanolamine and phosphatidylcholine had the greatest unstimulated metabolic incorporation rates and exhibited the greatest changes in incorporation rates in activated cells. High concentrations of concanavalin A (greater than 4 pg/ml) caused less stimulation of the am6unt of acetate incorporated into phosphatidylcholine when compared to lower mitogen concentrations, while phosphatidylethanolamine and triacylglycerol incorporation rates remained elevated at high concanavalin A concentrations. Because higher concentrations of concanavalin A appeared to interfere only with incorporation of [ 14C]acetate into phosphatidylcholine and not into phosphatidylethanolamine or triacylglycerol, the enzyme acyltransferase responsible

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F i g . 3. I n c o r p o r a t i o n of [ 14C]acetate into mouse splenic lymphocyte lipids as a function of concanavahn A concentration. Lymphocytes were incubated with the appropriate concentration of lectm and [ 14C]_ a c e t a t e ( 2 5 n m o l ; s p e c . a c t . o f 5 8 C i / m o l ) f o r 4 h a t 3 7 ° C . [ 14 C ] A c e t a t e i n c o r p o r a t i o n is e x p r e s s e d as t h e mean percentage of control values for three determinations. The individual lipids with control dpm values in parentheses are: m phosphatidylethanolamine (100% = 1046 + 108 dpm); • triacylgycerol (100% = 1 8 3 8 + 3 7 0 d p m ) ; *, p h o s p h a t i d y l c h o l i n e ( 1 0 0 % = 1 6 9 0 -+ 1 1 3 d p m ) ; o l y s o p h o s p h a t i d y l c h o l i n e (100% = 2 2 2 + 2 8 d p m ) ; ~ , f r e e f a t t y a c i d ( 1 0 0 % = 4 1 6 -+ 4 5 d p m ) . Fig. 4. The inhibition of acyltransferase activity m mouse splenic lymphocytes as a f u n c t i o n o f c o n c a n a valin A concentration. A c y l t r a n s f e r a s e a c t i v i t y w a s a s s a y e d in t h e i n t a c t cells a f t e r a 4 h i n c u b a t i o n w i t h t h e l e c t i n , a n d is e x p r e s s e d a s a p e r c e n t a g e o f t h e m e a n c o n t r o l v a l u e w h i c h w a s 0 . 8 5 +- 0 . 0 9 n m o l / m i n p e r 1 0 7 cells. E a c h p o i n t is t h e m e a n w i t h s t a n d a r d d e v i a t i o n f o r s e v e n a s s a y s . T h e s o l i d l i n e is f o r c o n c a n a v a l i n A a n d t h e b r o k e n l i n e is f o r s u c c i n y l a t e d c o n c a n a v a l i n A .

for the re-acylation of lysophosphatidylcholine, was examined 4 h after the addition o f the lectins to lymphocytes. Acyltransferase was inhibited by increasing concanavalin A concentrations up to a maximum of 60% inhibition (Fig. 4). Succinyl concanavalin A gave little or no inhibition of acyltransferase activity over the concentrations tested (10-6--10 -3 g/ml). Possible causes for concanavalin A inhibition and for lack of succinyl-concanavalin A inhibition of acyltransferase were investigated. Viabilities measured b y [SlCr]chromate release indicated that the t w o lectins were n o t toxic to the l y m p h o c y t e s in this short-term culture system (Viability was greater than 90% at 4 h for lectin concentrations of 0--100 pg/ml). Examination of l y m p h o c y t e suspensions b y light microscopy revealed that concanavalin A induced some l y m p h o c y t e agglutination at concentrations that inhibited acyltransferase activity (Table Ia). Since cellular agglutination might result in reduced access of substrate to the l y m p h o c y t e plasma membrane enzyme, the effect of agglutination on acyltransferase activity was examined. The inhibition of acyltransferase and l y m p h o c y t e agglutination could be spontaneously reversed by 0.1 M a-methylmannoside (Table Ia), a concentration which displaces most of the lectin from the l y m p h o c y t e surface [1,21]. In addition, succinyl concanavalin A did n o t cause agglutination or affect acyltransferase activity (Table Ib). However a number of lectins from sources such as Pisum sativum, Phaseolus vulgaris, Lens culinaris, and Ricinus communis were also observed to induce splenic l y m p h o c y t e agglutination, without inhibiting acyltransferase activity (Table Ic). Cross-linking of succinyl concanavalin A with anti-concanavalin A antibody, under appropriate conditions [22], caused l y m p h o c y t e agglutination without altering acyltransferase levels (Table Ib).

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TABLE I C O M P A R I S O N S O F T H E E F F E C T S O N L Y M P H O C Y T E A C Y L T R A N S F E R A S E A N D ON L Y M P H O C Y T E A G G L U T I N A T I O N O F C O N C A N A V A L I N A, S U C C I N Y L C O N C A N A V A L I N A, O T H E R LECTINS, ANTI-CONCANAVALIN-A SERUM, COLCHICINE AND LUMICOLCHINE A c y l t r a n s f e r a s e a c t i v i t y w a s m e a s u r e d a f t e r 4 h i n c u b a t i o n o f m o u s e spleen l y m p h o c y t e s w i t h t h e l e c t i n , a n d is e x p r e s s e d as a p e r c e n t a g e o f c o n t r o l s , the activity o f w h i c h w a s 0 . 8 8 ± 0 . 0 8 n m o l / m i n p e r 107 cells. 0.1 M a - m e t h y l m a n n o s i d e w a s a d d e d w h e r e indicated, 15 rain b e f o r e the a c y l t r a n s f e r a s e assay. A n t i - c o n canavaiin A serum containing 100 Dg/ml of anti-concanvaiin A a n t i b o d y , colchicine or lumicolehicine were present w h e r e indicated, for the last 2 h o f i n c u b a t i o n . All d e t e r m i n a t i o n s were d o n e in triplicate, a n d t h e m e a n w i t h s t a n d a r d d e v i a t i o n is s h o w n for a c y l t r a n s f e r a s e assays. A g g l u t i n a t i o n is e x p r e s s e d as the r a n g e o f t h e percentage o f total cells that were agglutinated, for three e s t i m a t e s . Acyltransferase (%)

Agglutination (%)

(a) C o n c a n a v a i i n A e f f e c t s Control ~-Methylmarmoside Coneanavaiin A (16 Dg/ml) C o n c a n a v a l i n A (16 D g / m l ) + ~ - M e t h y l m a n n o s i d e

100 102 ± 4 40 + 5 9 5 -+ 3

2--10 2--10 90--98 4--15

(b) S u c c i n y l c o n c a n a v a l i n A e f f e c t s Succinyl concanavaiin A (32/zg/ml) S u c c i n y l c o n c a n a v a l i n A (1 m g ] m l ) Anti-concanavalin A serum Succinyl concanavalin A (32/~g/ml + anti-concanavalin A serum S u c c i n y l c o n c a n a v a l i n A (1 r a g / m 1 ) + a n t i - c o n c a n a v a i i n A s e r u m

95 90 102 106 104

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3 6 4 7 3

6--12 20--25 2--7 70--80 90--94

(c) O t h e r l e c t i n e f f e c t s Phaseolus uulgaris ( 2 0 0 ~ug/mi) Pisurn s a t i u u m ( 2 0 0 / ~ g / m l ) Ricinu8 c o m m u n i $ ( 2 0 0 # g / m l ) L e n s cuUnaris ( 1 0 0 0 ~tg/ml)

99 80 94 110

±4 _+ 6 ± 3 ± 4

85--90 80--90 80--85 90--94

(d) C o l c h i c i n e e f f e c t s C o l c h i c i n e (10 - s M) C o n c a n a v a l i n A ( 1 6 / ~ g / m ] ) + c o l c h i c i n e ( 1 0 -5 M) C o n c a n a v a l i n A ( 1 6 / ~ g / m l ) + l u m i c o l c h i c i n e (10 -5 M)

102 ± 6 44 ± 5 38 ± 7

2--10 30--35 85--95

Furthermore colchicine, at concentrations which disrupt microtubules (10 -s M), caused a reversal of concanavalin A-induced lymphocyte agglutination but did not affect basal or concanavalin A-inhibited acyltransferase activity (Table Id). Lumicolchicine had no effect on concanavalin A-induced agglutination or inhibition of acyltransferase. One possible interpretation of the above results, based on literature reports of the hydrophobic properties of concanavalin A [17,19,23,24], is that the inhibition of acyltransferase by concanavalin A involves a lectin-induced hydrophobic perturbation of the lymphocyte plasma membrane. Since the binding o f concanavalin A to lipid vesicles has been used to evaluate its hydrophobic properties [17,23], the ability of succinyl concanavalin A and concanavalin A to bind and interact with dipalmitoylphosphatidylcholine vesicles was examined to determine their relative hydrophobicities. As shown in Fig. 5, the addition of concanavalin A to a unilamellar vesicle suspension resulted in an increase in turbidity of the suspension as monitored by the increase in absorbance at 450 nm. The increase in turbidity was dependent on the concentration of lectin (0.1--5 mg/ml). Succinyl concanavalin A (0.1--5 mg/ml) did not significantly alter turbidity

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F i g . 5. T h e a g g r e g a t i o n o f s o n i c a t e d vesicles o f d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e b y c o n c a n a v a l i n A. A g g r e g a t i o n w a s m e a s u r e d as t h e i n c r e a s e in t u r b i d i t y , in a b s o r b a n c e u n i t s , at 4 5 0 n m d u r i n g 3 h i n c u b a t i o n at 2 5 ° C . Solid lines r e p r e s e n t c o n c a n a v a h n A a d d i t i o n ( 0 . 1 - - 5 m g / m l ) a n d t h e b r o k e n line s u c c i n y l a t e d c o n c a n a v a l i n A (5 m g / m l ) , l o w e r c o n c e n t r a t i o n s o f t h e l a t t e r gave s i m i l a r c u r v e s t o t h e o n e s h o w n . E a c h v a l u e s h o w n is t h e m e a n w i t h s t a n d a r d d e v i a t i o n f o r t h r e e d e t e r m i n a t i o n s . F i g . 6. T h e e f f e c t s o f v a r i o u s c o n c e n t r a t i o n s o f c o n c a n a v a l i n A, a n d o f v a r i o u s t i m e s of e x p o s u r e to t h e l e c t i n , on [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n i n t o m o u s e s p l e n i c l y m p h o c y t e s . I n c o r p o r a t i o n o f [ 3 H ] t h y m i dine into trichloroacetic acid-insoluble material was m e a s u r e d for the period 4 8 - - 5 0 h after lectin addit i o n . 0.1 M a - m e t h y l m a n n o s i d e w a s a d d e d , at t h e t i m e s s t a t e d in t h e a b s c i s s a , t o d i s p l a c e b o u n d l e c t i n f r o m t h e cells. T h y m i d i n e u p t a k e u n i t s are c p m X 10 - 3 , t h e h o r i z o n t a l c r o s s - h a t c h e d b a r s are f o r c o n c a n a v a l i n A 1 /~g/ml; clear b a r s are c o n c a n a v a l i n A 4 p g / m l a n d d i a g o n a l l y c r o s s - h a t c h e d b a r s are c o n c a n a v a l i n A 16 p g / m l . E a c h value is t h e m e a n o f f o u r d e t e r m i n a t i o n s .

from control values. Precipitation of lipid vesicles was not observed at any concentration of lectin tested. Although a stoichiometric correlation between turbidity and lectin-vesicle interaction has n o t been established in these experiments, van der Bosch and McConnell [17] have presented electron micrography and ESR evidence to indicate that concanavalin A induces membrane perturbation of dipalmitoylphosphatidylcholine vesicles leading to vesicle fusion and aggregation. Such an aggregation would result in the turbidity changes observed here. a-Methylmannoside was used to study the effect on l y m p h o c y t e transformation of short exposures to concanavalin A. This glycoside was added to mouse spleen l y m p h o c y t e s at various times after the addition of the lectin, in order to reverse the binding of the bulk of the lectin to the cell surface, and to determine the time of exposure to lectin required,for the c o m m i t m e n t of the cells to divide. As shown in Fig. 6 the l y m p h o c y t e s required only 4 h exposure to concanavalin A to become c o m m i t t e d to divide, the mitogenesis being measured by [3H]thymidine incorporation 48 h after the addition of the lectin. During 4 h of exposure, the lymphocytes are equally committed to divide, whether t h e y are exposed to 1, 4 or 16 pg lectin per ml. Only if exposed for 8 h or more is there suboptimal mitogenesis at concanavalin A levels greater than I pg/ ml. This suggests t h a t the inhibitory phase of the mitogenic response to concanavalin A, seen at higher concanavalin A concentrations, is due to a long-term effect and is n o t dependent on the initial stimulus to divide. In particular l y m p h o c y t e s which have been exposed to concanavalin A, at a level of 16/~g/ ml for 4 h, display only 40% of the acyltransferase level of unstimulated cells

313 (Fig. 4) and yet they are as committed to divide as cells which have been exposed to the lectin at 1 #g/ml for the same period of time. Discussion

The lymphocyte plasma enzyme, acyltransferase, has been implicated in some models for lymphocyte activation [25--27]. The findings of the present study indicate that in intact mouse lymphocytes stimulated by concanavalin A there is no activation of acyltransferase at any lectin concentration tested. O n the contrary, there is inhibition of total acyltransferase activity in these cells by the lectin at and above optimal mitogenic concentrations. While the present study confirms the observations that there is a rapid stimulation of lipid precursors into phospholipid and triacylglycerol following exposure to lectins [28,29] it suggests that the stimulation of lipid synthesis is not due to acyltransferase activation. Our findings are in line with those of Toyoshima and Osawa who found no evidence for increased polyenoic fatty acid incorporation in h u m a n peripheral lymphocytes stimulated with the mitogenic lectins, Wistaria floribunda and L. culinaris [30,31]. Concentrations of concanavalin A that inhibit acyltransferase in this study are similar to those that block receptor mobility in human peripheral lymphocytes [32]. The profile of concanavalin A inhibition of acyltransferase at 4 h is very similar to the inhibitory phase of the biphasic effect of concanavalin A concentrations on thymidine incorporation at 48 h. McClain and Edelman have predicted that any measured biochemical result of lectin binding that shows a concanavalin A dose-response curve with an inhibitory phase at high lectin concentrations is probably a later event and is not in the initial stimulatory pathway of l y m p h o c y t e activation [1]. Inhibition of acyltransferase appears to occur in parallel with other inhibitory effects of concanavalin A. This is further evidence that stimulated acyltransferase activity is not required for l y m p h o c y t e activation. The inhibition of acyltransferase in intact mouse lymphocytes by concanavalin A is reversed by a-methylmannoside, which indicates that glycoside-specific binding of the lectin to the receptor is necessary, and that the inhibition is not a function of some unrelated minor lipophflic contaminant of the lectin. In agreement with Milner [33], we find that only 4 h exposure to concanavalin A is needed to achieve a substantial proportion of the maximal degree of l y m p h o c y t e transformation. More importantly, this transformation by 4 h exposure to lectin is achieved at concanavalin A concentrations, i.e. 16/~g/ml, which will inhibit 60% of total acyltransferase activity. Thus the cells become committed to divide while most of their acyltransferase activity is inhibited. Concanavalin A-induced lymphocyte agglutination was also spontaneously reversed by the addition of ~-methylmannoside. However a n u m b e r of experiments suggest that agglutination is not the mechanism by which the enzyme is inhibited by the lectin. Other lectins which could induce agglutination did so without affecting acyltransferase activity. These included lectins with similar glycoprotein-receptor specificities to concanavalin A (P. sativurn, L. culinaris) and lectins with different specificites (P. vulgaris, R. communis). In addition, cross-linking of the normally non-inhibiting and non-agglutinating succinyl

314 concanavalin A with anti~oncanavalin A antibody induced significant agglutination without altering acyltransferase activity. Interestingly colchicine, at concentrations which disrupt microtubules ( 10- s M) [ 34 ], did n o t affect the inhibition of acyltransferase by concanavalin A, b u t did reverse agglutination. The effect of colchicine on reversal of concanavalin A-induced agglutination has been reported in other cell types [35--37]. Ferber, Reilly and Resch found colchicine to be without effect on the concanavalin A-induced activation of acyltransferase activity in calf thymocyte plasma membrane fragments [38]. While our results suggest that colchicine-binding proteins are n o t involved in the inhibition o f acyltransferase, it would be premature to conclude that the cytoskeleton is unaffected by high concentrations of concanavalin A. The effect of lectin-receptor cross-linking on acyltransferase activity was examined as a possible mechanism b y which the enzyme was inhibited. Concanavalin A, a tetravalent ligand which causes extensive plasma membrane glycoprotein cross-linking, inhibited acyltransferase; while succinyl concanavalin A, a divalent ligand that induces less cross-linking [22], does not inhibit acyltransferase. Cross-linking succinyl concanavalin A with a divalent antibody, at concentrations (100 pg/ml) that cause succinyl concanavalin A to inhibit membrane receptor m o v e m e n t [37] and induce l y m p h o c y t e agglutination in a similar manner as high concanavalin A concentrations, did n o t affect the activity of acyltransferase. This suggests that although cross-linking of membrane receptors for concanavalin A may contribute to agglutination and receptor mobility inhibition, it is n o t required for acyltransferase inhibition. However, our experiments do n o t rule out the possibility that cross-linking by concanavalin A is required for inhibition of the enzyme, while cross-linking b y the antibody under our conditions is insufficient for inhibition. On the basis of the present study we suggest that concanavalin A inhibition of acyltransferase is n o t due to toxicity, agglutination, direct cross-linking through carbohydrate-containing surface receptors or indirect cross-linking though colchicine-binding proteins (microtubules). According to Edelman's hypotheses concerning concanavalin A-induced inhibition mechanisms there is one remaining possibility, that is, the inhibition of acyltransferase could be due to a direct perturbation of the plasma membrane lipid bilayer by the lectin [19]. Data suggesting that this is a possible mechanism of inhibition include: evidence of a h y d r o p h o b i c region in the concanavalin A molecule; identification of cell surface-adsorption n o t involving a specific carbohydrate receptor; and evidence for direct fluidity changes in the plasma membrane induced b y other lectins. We have previously shown that mouse l y m p h o c y t e acyltransferase is sensitive to inhibition b y micromolar concentrations of hydrophobic molecules such as retinol and cannabinoids [16]. Other plasma membrane enzymes, e.g. 5'nucleotidase, are known to be inhibited b y concanavalin A [39]. It may be that the activation b y concanavalin A of acyltransferase observed b y Resch and co-workers in membrane fragments is actually a lifting of inhibition induced during isolation of membranes by lipophilic products of autolysis, notably lysophosphatidylcholine or free fatty acids, both of which will inhibit acyltransferase.

315

Prewous literature reports have indicated that concanavalin A can interact directly with hydrophobic molecules including lipid vesicles lacking any carbohydrate residues [17,19]. We have compared the aggregation of synthetic unilamellar dipalmitoylphosphatidylcholine vesicles induced by concanavalin A and succinyl concanavalin A. It is clear that concanavalin A is a much better vesicle-aggregating agent than succinyl concanavalin A. Van der Bosch and McConnell have suggested that this vesicle aggregation is the result of a hydrophobic-like perturbation of the lipid bilayer that facilitates membrane fusion [17]. The addition of ten succinyl groups to each concanavalin A monomer could prevent vesicle aggregation by imparting considerable polarity to the surface of succinyl concanavalin A. If the ability of concanavalin A to aggregate vesicles through membrane perturbation is an appropriate model for the perturbation of the lymphocyte membrane by concanavalin A, then differences in vesicle membrane perturbation between these two lectins are consistent with their ability to alter the activity of the lymphocyte membrane enzyme, acyltransferase. In light of our other data, this would suggest that the inhibition of acyltransferase by concanavalin A is the result of hydrophobic interaction between the lectin membrane components. Acknowledgements This work was supported by N.S.E.R.C. Canada and by M.R.C. Canada. The authors are grateful to Dr. R.A.B. Keates and Dr. B.N. Wilkie for useful discussions. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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