THE JOURNAL. OF BIOLOGICAL CHEMISTRY Vol. 251. No. 7,lssue of April 10, pp. 1864-1870, 1976

Printed in U.S.A.

Surf’ace Alterations in Calf’ Lymphocytes Oxidized by Sodium Periodate*

(Received l’or publication, September 10. 1975)

CARY A. ~RESANT$ AND SUSAN PARKER

From the Division of Hematology and Oncology, Department of Medicine, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, Missouri 63110

In order to investigate alterations in surface structure in transformed lymphocytes, calf submandibular

lymph node cell suspensions were oxidized with NaIO,. Oxidized lymphocytes were morphologically

transformed and had higher rates of DNA synthesis by 2 days after treatment. These results were

prevented by reduction of the cell suspension with h’aBH,, or by neuraminidase treatment of cells prior

to oxidation.

The amount of ‘251-labeled Agaricus bisporus lectin bound to cells immediately after oxidation and the

affinity constant for binding were increased over 2 -fold, while cells immediately following oxidation and

reduction showed decreased receptors with still higher affinity for the lectin compared to untreated cells.

The amount of Phoseolus oulgaris lectin bound to oxidized cells was also increased, but affinity was

unchanged. Immediately following oxidation and reduction, these receptor sites were unchanged in

number and affinity from untreated cells. In contrast, the number and affinity of receptors for

concanavalin A were not changed immediately after oxidation or oxidation and reduction.

In order to define the extent of compositional changes in surface glycoprotein receptors, plasma

membranes were isolated from frozen calf submandibular lymph nodes. Compared to untreated plasma

membranes, oxidized membranes had similar contents of galactose, mannose, N-acetylglucosamine,

N-acetylgalactosamine, fucose, and amino acids. Sialic acid content of oxidized membranes was reduced

when measured by thiobarbituric acid assay. Sialic acids of untreated plasma membranes co-chromato-

graphed with N-glycolylneuraminic acid and N-acetylneuraminic acid, while those of oxidized

membranes co-chromatographed with N-glycolylneuraminic acid and 5-acetamido-3.5-dideoxy-L-

arabino-7-aldehydo-2-heptulosonic acid.

Therefore, specific surface conformational changes in certain classes of membrane glgcoproteins are

associated with mild Malapradian oxidation of membrane sialic acids. These temporally precede

NaIO,-induced transformation of calf lymphocytes. This is consistent with an hypothesis of membrane-

mediated stimulation of lymphocyte transformation.

Initiation of cellular proliferation has been studied in lym-

phocytes transformed by a variety of stimuli, such as soluble

antigens, mitogenic plant lectins, or histoincompatible lym-

phocytes. Recently, it has been shown that oxidation either by

NaIO, (1,2) or by neuraminidase plus galactose oxidase (3) can

also increase DNA synthesis in cultures of mouse, rat, or

human lymphocytes. Evidence suggests that transformation of

mouse lymphocytes by NaIO, is specific for thymus-dependent

* This work was supported in part by United States Public Health Service Grant CA 15621 from the Division of Cancer Research, Resources, and Centers of the National Cancer Institute; American Cancer Society Grant BCl55; and United States Public Health Service General Research Support Grant CA 10435. This work was presented in part at the Ninth Leukocyte Culture Conference, Williamsburg, Virginia. December 19i4.

$ To whom reprint requests should be addressed: Cary A. Presant, M.D., Division of Hematology, The Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis. MO. 63110.

lymphocytes (4). It has been proposed that both methods of

oxidation (3, 5) result in transformation of the oxidized

lymphocyte by formation of aldehydes from sialic acid or

galactose, and that such aldehydes cross-link with other

surface structures (perhaps free amino groups) to form a

surface “lattice.”

Lymphocytes that have been transformed by NaIO, provide

an excellent system for investigating alterations in surface

structure, since the cells have not been coated by foreign

proteins such as lectins or antigens. Therefore, this investiga-

tion examined changes in surface structure of calf submandib-

ular lymph node lymphocytes resulting from periodate oxida-

tion, or oxidation plus NaBH, reduction.

EXPERIMENTAL PROCEDURE

Materials-Concanavalin A was purchased tram Miles-Yeda (Kan- kakee, Ill.) and was extensively dialyzed against 0.15 M NaCl prior to

1864

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Surface Alterations in Oxidized L.ymphocytes 1865

use. The erythroagglutinating lectin of Phaseolus vulgaris (E-PHA)’ was purified from Bacto-phytohemagglutinin-P (Difco, Detroit, Mich.) by the method of Weber et al. (6) to apparent homogeneity as determined by polyacrylamide gel electrophoresis. Agaricus bisporus, commercial mushrooms (Moonlight Mushrooms, Butler County Farm, West Winfield, Pa.) were purchased in a local supermarket. One of the lectins (mushroom PHA-B) was purified from these mushrooms by the method of Presant and Kornfeld (7). Tritiated thymidine and NaBSH, were obtained from New England Nuclear (Boston, Mass.) and Na’151 was purchased from Amersham/Searle (Arlington Heights, Ill.). Fetal calf serum and Medium 199 were obtained from Grand Island Biological Co. (Grand Island, New York). N-Acetylneuraminic acid and N-glycolylneuraminic acid were purchased as standards for assay and chromatography from Sigma (St. Louis, MO.). Vibrio cholera neuraminidase was purchased from Calbiochem (La Jolla, Calif.).

Lymphocyte Suspensions-Fresh calf submandibular nodes were donated by Star Meat Packing Co. (St. Louis, MO.). The lymph nodes were dissected free of connective tissue, washed in sterile 0.15 M NaCl, and then minced in a small volume of 0.15 M NaCl. After large tissue clumps had settled, lymphocytes in the supernatant fluid were centrifuged and then resuspended in 0.15 M NaCl. Binding studies were performed immediately after resuspension in 0.15 M NaCl.

Periodate oxidation was performed by incubating lymphocytes at a concentration of 20 x 10’ cells/ml in 0.4 mM NaIOJ0.005 M sodium phosphate, pH 7.4/0.15 M NaCl for 15 min at 25”. The reaction was terminated by centrifugation and resuspension twice in Medium 199, and then in 0.15 M NaCl. Binding studies were performed immediately after suspension in 0.15 M NaCl.

Periodate-oxidized lymphocytes were reduced in sodium boro- hydride by incubation at a concentration of 20 x 10’ cells/ml in freshly prepared 1 rnhr NaBH,/0.005 M sodium phosphate, pH 7.4/0.15 M NaCl for 15 min at 25” in a lighted laboratory. This reaction was terminated by centrifugation and resuspension of the pellet twice in 0.15 M NaCl. Binding studies were performed immediately after the final suspension in 0.15 M NaCl.

Binding Assays-Lectins were iodinated with ‘*? by the method of Hunter (8) employing a 5-s exposure to chloramine T. The ability of the lectins to agglutinate cells was unchanged after this procedure. Over 90% of the radioactivity in each lectin could he bound to an excess of cells.

Binding assays were performed as previously described (7). Plastic tubes were incubated for 60 min with 1 ml of 5 mg/ml of bovine serum albumin in 0.15 M NaCl/lO mM NaHCO,, and then aspirated. Reaction mixtures contained 2 x lo6 untreated, oxidized, or oxidized and reduced calf lymphocytes immediately after suspension in 0.05 ml of 0.15 M NaCI: 0.1 ml of bovine serum albumin 5 mg/ml in 0.15 M NaCl/lO mM NaHCO,; and 0.05 ml of ‘zSI-labeled lectin. After incubation at 25” for 30 min with periodic shaking, the mixture was centrifuged and the pellet was washed three times with 5 ml of 0.15 M NaCl. Cell-associated radioactivity was then determined using a Nuclear-Chicago model 4223 gamma counting system. Corrections were made for nonspecific bindin g by including reaction mixtures lacking cells in the case of P. vulgaris and mushroom PHA-B, and reaction mixtures with 0.4 M a-methyl glucose in the case of Con A. Nonspecific lectin binding, which amounted to about 5’; ofexperimen- tal values for mushroom PHA-B, 15’~ for P. vulgaris E-PHA, and 35% for Con A, was subtracted in each instance. If mixtures containing cells plus fetuin (0.5 mg) were used as controls for nonspecific binding (instead of mixtures without cells), specific binding of mushroom PHA-B was 3.8’:< less, and of P. vulgaris E-PHA was 14% less. All binding studies were performed in duplicate. Binding of all lectins was 96% complete in 30 min.

Data were examined by two methods. Conformity to first order kinetics (or one class of receptor sites) was established by Scatchard plots (9) as discussed b\, Walter (10). The data were also plotted by the method of Steck and U’allach (ll), and the number of binding sites per cell and the association constant for each lectin were calculated by linear regression analysis of points for each experiment. Regression coefficients for data in each experiment ranged from 0.95 to 0.99.

Chemical Assays-Protein was measured by the method of Lowry et al. (1%). DNA was assayed by the diphenylamine reaction of Burton (13) following hydrolysis of samples in 7.5’~ perchloric acid at 70” for

’ The abbreviations used are: PHA, phytohemagglutinin; Con A, concanavalin A; Neuh’Ac-7. 5-acetamido-3,5-dideoxy-L-arabino-2. heptulosonic acid; NeuNAc-7.ald, 5.acetamido-:1,5-dide[)x~-L- arabino-7.aldehydw2.heptulosonic acid.

15 min. Amino acid analysis was performed using a Spinco automatic amino acid analyzer on samples hydrolyzed in constant boiling HCI for 16 hours at 108” in an evacuated sealed tube. Free sialic acid was measured by the method of Warren (14) with hydrolysis of sample in 0.05 N H,SO. at 80’ for 1 hour to release terminal sialic acid when indicated. N-Acetylneuraminic acid was used as the standard. Galac- tose and mannose were measured enzymatically following sample hydrolysis in 2 N H,SO, for 4 hours at 100” and deionization on Amberlite MB3, as described by Kornfeld et al. (15). Glucosamine and galactosamine were quantitated on a Spinco automatic amino acid analyzer following hydrolysis in 4 N HCI for 4 hours at 100” in uacuo. Total hexosamine was determined after similar hydrolysis and adsorp- tion to and elution from Dowex 50, by the method of Reissig et al. (16). Fucose was assayed by the cysteine-H,SO, method (17).

5’-Nucleotidase was measured by the method of Weaver and Boyle (18) as a marker for plasma membrane. @-N-Acetylglucosaminidase was measured as described (19) as a marker for lysosomes. Succinate dehydrogenase was assayed by the method of Earl and Korner (20) as a marker for mitochondria.

Lymphocyte Cultures-Short term cultures of various lymphocyte suspensions were established as previously described (7). Each culture tube contained 2 ml of Medium 199 with 10% fetal calf serum. 4 mmol of glutamine, 100 units of penicillin, 100 fig of streptomycin, and 4 to 8 x 10’ calf lymphocytes. After incubation for 48 hours at 37" in 5% CO,/95% air, 3 &i of [‘Hlthymidine was added to each culture tube for 4 hours. The cells were washed with 9 ml of cold 0.15 M NaCl, suspended in 5 ml of cold 5’i, trichloroacetic acid. and sonicated. After centrifugation at 12,000 x g for 10 min, pellets were washed once with 8 ml of cold trichloroacetic acid, dissolved in 0.5 ml of NCS solubilizer, and counted in Bray’s solution in a Packard Tri-Carb scintillation spectrometer.

Preparation of Calf L,ymph Node Plasma Membranes-A method was developed in association with Kornfeld and Siemers (19). Large quantities i200 to 1.000 g) of frozen calf submandibular lymph nodes were obtained from Pel-Freeze (Rogers, Ark.) or from Star Meat Packing Co. (St. Louis, MO.). These were passed through a high speed tissue shredder (Hobart model 4612 power unit equipped with a Hogo 3:l speed attachment and g-inch vegetable shredder plate. Hobart Manufacturing Co.. Troy, Ohio). Shredded tissue was collected in chilled 0.25 M sucrose/50 rnM Tris/l mM EDTA, pH 7.5, and was homogenized for 10 s in a Waring Blendor. The homogenate was filtered through cheesecloth and the filtrate was centrifuged at 980 x g for 10 min to remove nuclei and intact cells. The supernatant solution was brought to 0.1 M LiCl by addition of 3 M LiCl. and was then centrifuged at 4000 x g for 15 min. The pellet contained mitochondria and was discarded. The supernatant solution was centrifuged at 50.000 x g at 4’ for 1 hour; the 50,000 x g supernatant solution containing rihosomes was discarded. and the 50.000 x g pellet was dispersed in sucrose/l0 mM Tris. pH 7.5, to a.f’inal concentration of 40’, sucrose (w/w) hy homogenization in a 50.ml homogenizer (Kontes Glass Co.). This was distributed into cellulose nitrate tuhes (18 ml/tube) and carefully layered over with 30’~ (w/w) sucrose in 10 mM Tris, pH 7.5 (18.5 ml/tube). The tubes were centrifuged for 4 hours at 7:1,000 \. g in a Spinco model 30 rotor in a Beckman ultracentrifuge. Four fractions were obtained by aspiration: a cloudy supernatant solution (“upper”). a dense milky band at the interface (“middle”). a clear solution below the interface (“bottom”). and a pellet. These fractions were each centrifuged at 50,000 x g at 4” for 1 hour, and the pellets were washed twice with 10 rnM Tris, pH 7.5. and stored frozen until use.

Fractions thus ohtained were assayed for marker enzymes, DSA. protein, and sialic acid (Table I). This indicated that the upper fraction consisted of plasma membranes plus a small amount of lysosomes, the middle was composed of plasma membranes, lyso- somes, and mitochondria. and the pellet contained DNA, mitochon- dria, lysosomes, and plasma membranes. Electron microscopy per- formed on the upper and middle fractions was consistent with the biochemical analysis. While the upper fraction consisted almost entirely of plasma membranes. the middle fraction also contained a large amount of mitochondria. Therefore, the upper fraction was used for subsequent biochemical analyses.

RESULTS

Characterization of Calf Lymphocyte System-Cell suspen- sions were made f’rom fresh calf submandibular lymph nodes. Untreated cell suspensions contained 875 small, 9.5% me- dium, and 2% large lymphocytes, no lymphoblasts, and 1.5%

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1866 Surface Alterations in Oxidized Lymphocytes

TABLE I

Characterization of lymphocyte subcellular fractions

Calf submandibular nodes were processed and assayed as described in the text. Crude homogenate is the supernatant solution after the 980 x g centrifugation step (see under “Experimental Procedure”). After centrifugation of the crude homogenate at 4,000 x g, the pellet was discarded; and after centrifugation at 50,000 x g, the supernatant solution was discarded before applying the pellet to the gradient indicated. % refers to percentage of the amount in the crude homogenate.

Fraction Protein Sialic acid 5’.Nucleotidase Succinic dehydrogenase

0.N-Acetglglu cosaminidase

mg ‘( rmol ‘i nmoll

w’ w T < “ZRhR Ub f% UhR U % ulmz u 9 lhR

Crude 7000 loo 53.5 100 8 1130 100 0.16 4500 100 0.64 1360 100 0.19 4630 100 0.67 Homogenate

Gradient Upper 71 1.0 3.6 6.7 51 1.5 0.14 0.022 1460 32.5 20.6 1.5 0.11 0.02 20 1 4.6 2.9 Middle 540 7.7 11.8 21.0 22 2.3 0.20 0.004 1020 22.7 1.9 251 18.5 0.47 1240 27.0 2.3 Bottom 79 1.1 0.5 0.9 6 0.8 0.07 0.010 115 2.6 1.5 84 6.2 1.07 306 6.8 4.0 Pellet 1308 18.7 9.6 18.0 7 682 60.0 0.52 1140 25.4 0.9 1370 100 1.05 642 14.0 0.5

o Specific activity in nmol/mg of protein. b U, units or micromoles of substrate converted per hour.

monocytes. After oxidation in NaIO,, the cell suspension consisted of 82% small, 13% medium, and 2% large Iympho- cytes, no lymphoblasts, and 3% monocytes. The cells were 99% viable both before and after oxidation as determined by trypan blue exclusion, and 88% viable after NaBH, reduction. After 2 days in short term culture, lymphoblasts accounted for 2.1% + 0.6% (1 S.E.) of cells in the untreated cell suspension, 21.3% + 1.8% of cells in the oxidized cell suspension, and 4.8% l 1.0% of cells in the oxidized and reduced cell suspension.

DNA synthesis was measured by tritiated thymidine incor- poration into cultures of calf lymphocytes (Table II). Addition to the cultures of an optimal amount of Phaseolus uulgaris

E-PHA, 15 fig/ml, or NaIO, oxidation of the cells resulted in an increased rate of DNA synthesis which was maximal at 48 hours. When NaIO, treatment was followed by NaBH, reduc- tion, the rate of DNA synthesis was equal to that in untreated cells. NaBH, treatment alone resulted in the same rate of DNA synthesis as in untreated cells.

Lectin Binding to Lymphocytes-Untreated, oxidized, and oxidized and reduced calf lymphocyte suspensions immedi- ately after preparation were tested for their ability to bind ‘Y-mushroom PHA-B. One experiment is illustrated (Fig. 1). Oxidized cells bound more radioactive lectin than untreated cells. Oxidation plus reduction did not increase the lectin binding to cells. Although increased affinity of the lectin for the cells was more evident in the replicate experiments, apparent affinity of’ the lectin for the cells was slightly increased after

oxidation, and further increased after oxidation plus reduction in the experiment illustrated (Fig. 1). Scatchard analyses of each of the three curves in this and other experiments with mushroom PHA-B indicated only one class of receptor (data not shown). This is also apparent in the double reciprocal plot (Fig. 1, right).

Results of five such binding studies are summarized in Table III. Oxidized cells had 2.6-fold more available receptor sites for mushroom PHA-B than untreated cells (p < 0.05). Lympho-

cytes which had been oxidized and reduced bound one-third less lectin than untreated cells (0.05 < p < 0.1). Affinity of

mushroom PHA-B for receptor sites was enhanced 2.4.fold by

oxidation of the cells (p < 0.05) and 5.6-fold by oxidation plus reduction (p < 0.05) compared to untreated cells.

A similar binding study using P. vulgaris E-““I-PHA also

suggested increased lectin binding to oxidized cells (Fig. 2).

TABLE II

DNA synthesis in calf lymphocytes Short term lymphocyte cultures contained 2 x lo* cells/ml. Cells

which had received no treatment were compared with cells cultured with Phaseolus oulgaris E-PHA, 15 rg/ml, cells which had been oxidized with h’aIO,, and cells which had been oxidized with h’aI0, and then reduced with hTaBH,. The rate of DNA synthesis was measured as the 4-hour incorporation of tritiated thymidine into acid-insoluble material at 48 hours. Stimulation index was calculated as the ratio of DNA synthesis rate in treated cells to that in untreated cells. The mean stimulation index L 1 S.E. and range of cpm per culture in 13 experiments are presented.

Treatment Stimulation index

None 1.0 hTaI0, 19.4 * 4.7 XaIO, + NaBH, 1.6 i 0.5 P. vulgaris E-PHA 10.6 * 2.7

cpm/Culture

146-261Z

16:W2”“28 144-1’90

16:16-l’iO”5

The binding of lectin to cells that had been oxidized and reduced was similar to that with untreated cells. The slight difference in association constant suggested in Fig. 2, right

panel, was not a consistent finding. Scatchard analyses demon- strated only one class of P. vulgaris E-PHA receptor for each of the three cell suspensions.

Data from four such P. vulgaris E-PHA binding experiments (Table III) indicated a 1.8-fold increase in available binding sites on lymphocytes following oxidation (p < 0.05). Although cells that had been oxidized and reduced had an average of one-third less receptor sites than untreated cells, this was not statistically significant (p > 0.4). Association constant deter- minations were more variable, and the average 2-fold differ- ence after oxidation and a l.‘i-fold decrease after oxidation plus reduction were not statistically different from untreated cells (p > 0.4).

In contrast to the results with mushroom PHA-B and P.

vulgaris E-PHA, the binding ot 1251-Con A to lymphocytes was slightly decreased after either oxidation, or oxidation plus reduction (Fig. 3 and Table III). The differences were small, however, and were not statistically significant (p > 0.4) due to greater variability in quantitative results. Affinity of Con A for the receptor site was similar in the three cell populations (p >

0.4). Scatchard analyses again demonstrated only one class of receptor.

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Surface Alterations in Oxidized Lymphocytes 1867

2 4 6 8 10 12 M-PHA-6 ADDED (~0)

TABLE III

Lectin binding to calf lymphocytes

Binding studies were performed as described under “Experimental Procedure” using ““I-labeled mushroom PHA-B. Phaseolus uulgaris E-PHA or Con A, and untreated. NaIO,-oxidized, or oxidized and NaBH,-reduced calf lymphocytes immediately after treatment. Means of five experiments (mushroom PHA-B) or four experiments (P. uulgaris E-PHA, Con A) are presented.

Cell Treatment

Lertin CJII- treated

Oxida- Oxidation

tion and reduction

Mushroom PHA-B

P. vulgaris E-PHA

Con A

Sites’ 1.42 3.62* 0.94’

K.Sd 3.59 8.54” 20.0’

Sites 2.17 3.97 1.36 K. 2.45 1.23 1.47

Sites 7.32 5.79 5.52 K. 0.15 0.16 0.15

o Sites x 10Ycell. bO.O25 < p < 0.05 compared to untreated cells. to.05 < p < 0.1. d K,, association constant, x lo* M-‘.

FIG. 2. Binding of Phaseolus uulgaris E-PHA to calf lymphocytes. Binding studies were performed as described under “Experimental Procedure.” Lymphocytes used were: untreated (O- - -01, oxidized with NaIO, (O-O), or oxidized and reduced with NaBH, (A- --A) immediately after preparation. This represents one experiment of a total of four summarized in Table II. Each point is the mean of duplicate determinations. Left, standard plot. Right, double reciprocal plot.

Composition of Calf Lymphocyte Plasma Membmnes- Purified calf lymphocyte plasma membrane suspensions were oxidized using the same conditions as described for calf lymphocyte suspensions. To each 10 mg of membrane protein

Y /

/

a2a40608 IO 12 11 16 Ifl

&- bJ"J

FIG. 1. Binding of mushroom PHA-B to calf lymphocytes. Binding studies were performed as described under “Ex- perimental Procedure.” Lymphocytes used were: untreated (O-- -01, oxi- dized with SaIO, (04). or oxidized and reduced with NaBH, (A---Al im- mediately after preparation. This repre- sents one experiment of a total of five summarized in Table II. Each point is the mean of duplicate determinations. Left, standard plot. Right, double recip- rocal plot. M-PHA-B, mushroom PHA-B.

FIG. 3. Binding Con A to calf lymphocytes. Binding studies were performed as described under “Experimental Procedure.” Lympho- cytes used were: untreated (O- - -0). oxidized with NaIO, (04). or oxidized and reduced with NaBH, (A---A) immediately after preparation. This represents one experiment of a total of four summa- rized in Table II. Each point is the mean of duplicate determinations. Left, standard plot. Right, double reciprocal plot.

(Lowry) was added 5 ml of 3.7 x lo-’ M NaI0,/0.005 M sodium phosphate, pH 7.4/0.15 M NaCl for 15 min at 25” in light, and the membranes were then centrifuged and washed with the same buffer without NaIO,. The membranes were then hydro- lyzed in conditions appropriate for the assay of amino acids or sugars as described under “Experimental Procedure.”

Sugar composition of the untreated and oxidized plasma membranes was similar except for sialic acid (Table IV). The thiobarbituric acid assay revealed only 69% of the original activity in oxidized membranes. Since mild oxidation of N-acetylneuraminic acid yields a product NeuNAc-7-ald which reacts about half as well in the thiobarbituric acid assay (21), the results suggested incomplete oxidation (about 60%) of sialic acid in lymphocyte plasma membranes to aldehydes. Contents of other sugars were not significantly different in oxidized membranes, and there was no loss of fucose content with oxidation.

Amino acid analysis showed no significant degradation of amino acids by oxidation (Table V). In particular, amino acids sensitive to non-Malapradian oxidation, threonine, serine, proline, cystine, and methionine, were unchanged within experimental error.

Sialic Acid Chromatography-Since the only apparent change in oxidized membranes was a decrease in thiobarbituric acid-assayable material, chromatography was performed to more precisely determine quantitative and qualitative aspects of the oxidation. Normal membranes, oxidized membranes, and oxidized membranes reduced by NaBaH, were each hydro- lyzed in dilute acid. The hydrolysate after centrifugation was subjected to descending paper chromatography in various solvents (Table VI). Migration of sugars was quantitated by

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1868 Surface Alterations in Oxidized Lymphocytes

TABLE IV TABLE VI

Calf lymphocyte plasma membrane composition

Untreated lymphocyte plasma membranes or h’aIO,-oxidized mem- branes were analyzed for sugar content. Assays were performed in duplicate for each plasma membrane preparation, and two plasma membrane preparations were analyzed separately. Values reported are the means of the two experiments. Content is expressed as Fmol of particular sugar/my of protein (Lowry). The molar ratio assumes the mannose content equal to 1.0.

Chromatographic behauior of sialic acids

Normal plasma membranes, oxidized membranes, and oxidized membranes reduced in 1W3hl NaBaH, for 10 min were each hydrolyzed in 0.05 N H,SO, at 80” for 60 min, and then brought to pH 7.0 with BaCO,. The supernatant solutions, following centrifugation twice at 50,000 x g for 60 min, were lyophilized. A portion of each equal to the hgdrolysate from 1.0 mg of membrane protein, or a sample of the standards or synthesized derivatives, wah subjected to descending chromatography on Whatman 3MM paper at room temperature. Solvent I, n-butyl acetate/acetic acid/water (3/l/11; Solvent Il. n-butyl alcohol/n-propyl alcohol/O.1 N HCl (111/l); Solvent III, n-butyl al- cohol/acetic acid/water (5/2/2). The paper was dried and stained with thiobarbituric acid (22) or cut into horizontal strips, eluted for 18 hours into water, lyophilized, and quantitated in a liquid scintillation counter.

Content Un- OX-

treated idized

Molar Ratio Oxidized, untreated IJn- OX-

treated idized

rmolfmn w

Sialic acids” 0.048 0.033 69 0.60 0.38

Mannose 0.080 0.088 110 1.0 1.0

Galactohe 0.151 0.154 10% 1.89 1.75 Fucose 0.069 0.079 < 106 0.86 0.83 Glucosamine 0.273 0.275 101 3.4 3.3

Galactosamine 0.064 0.064 loo 0.80 0.73

a Sialic acids are thiobarbituric acid-assayable material using N- acetylneuraminic acid as the standard.

TABLE V

Amino acid composition of calf lymphocyte plasma membranes

Untreated or NaIO,-oxidized calf lymphocyte plasma membranes were hydrolyzed as described under “Experimental Procedure” and assayed for amino acid content. Results are expressed as molecules of a particular amino acid/1000 amino acid molecules (residues/lOOO). The per cent change after oxidation is compared to untreated plasma membranes.

Amino acid

Residues/1000

Untreated Oxidized membranes membranes

Oxidized/ untreated

ratio

Aspartic acid 117 120 1.03 Threonine 60 65 1.08 Serine 112 112 1.00

Glutamine 151 135 0.89

Proline 61 66 1.08

Glycine 105 109 1.04

Alanine 83 89 1.07 Cystine 20 18 0.90 Valine 64 72 1.12 Methionine 15 13 0.87

Isoleucine 21 21 1.00

Leucine 82 70 0.85 Tyrosine 37 32 0.86

Phenylalanine 71 78 1.10

thiobarbituric acid stain (22) or liquid scintillation counting.

Standards used included N-acetylneuraminic acid and

N-glycolylneuraminic acid; NeuNAc-7; and NeuNAc-7-ald.

The latter two were synthesized as described by Liao et al. (21).

In addition, NeuNAc-7 generously provided by Dr. 0. 0.

Blumenfeld was included. The relative mobility of these

standards in the solvent systems employed are shown in Table

VI. The relative migration rates of N-acetylneuraminic acid

and N-glycolylneuraminic acid agree with previously pub- lished results in Solvent II (23).

Hydrolysate from untreated and oxidized plasma mem-

branes each showed two pink spots after staining the chro-

matograms from Solvents I or III with thiobarbituric acid.

Untreated plasma membrane hydrolysates co-chromato- graphed with N-glycolylneuraminic acid and N-acetyl-

Substance applied Mobility in solvent

I II III

Standard N-acetylneuraminic acid 1.0 1.0 1.0 (Sigma)

Synthetic NeuNAc-7-ald 1.82 1.52 1.4” Synthetic NeuNAc-7 1.82 1.51 N.T.

Synthetic SeuNAc-7 1.70 1.48 1.39 (Blumenfeld)a

Standard N-glycolylneuraminic 0.54 0.Z 0.5; acid (Sigma)

Untreated membrane hydrolysate 0.71.0.99 0.67 0.62, 1.08 Oxidized membrane hydrolysate 0.71, 1.76 0.69 0.64, 1.42 Oxidized and reduced membrane 0.54, 1.90 N.T.’ 0.63

hydrolysate*

a Mobility relative to N-acetylneuraminic acid. ‘Assayed by migration of tritium label. c N.T., not tested.

neuraminic acid. Oxidized plasma membrane hydrolysates

co-chromatographed with N-glycolylneuraminic acid, Neu- NAc-7-ald, and NeuNAc-5. Hydrolysates from reduced plasma

membranes assayed by tritium migration co-chromatographed with N-glycolylneuraminic acid, accounting for 93% of the radioactivity, and with NeuNAc-7 and NeuNAc-7-ald, ac- counting for only 7% of the radioactivity. No oxidized or

oxidized and reduced membrane hydrolysate co-chromato- graphed with N-acetylneuraminic acid. Conversely, no un- treated membrane hydrolysate co-chromatographed with Neu- NAc-7 or NeuNAcG-ald.

Neuraminidase Treatment of Lymphocytes-Calf Iympho- cytes were pretreated with Vibrio cholera neuraminidase

(Table VII). This enzyme was active, as measured by its ability to remove 0.79 + 0.1 rmol of sialic acid (measured as N-acetylneuraminic acid) from 10” calf lymphocytes (mean * 1 SE. for four experiments) or 0.63 rmol of sialic acid from lOLo human erythrocytes (one experiment) in 90 min at 37”.’ Sonicated calf lymphocytes hydrolyzed in 0.05 N H,SO, at 80” for 1 hour contained 1.2 pmol of free sialic acid per 10” ceils. Thus, neuraminidase was able to remove 66% of the sialic acid. Lymphocytes which had been treated with neuraminidase prior to NaIO, oxidation exhibited slightly decreased rates of DNA synthesis compared to untreated cells. Neuraminidase treatment did not decrease the stimulation of DNA synthesis by P. uulgaris, indicating that its effect on NaIO,-oxidized cells was not a consequence of general inhibition of DNA synthesis. Thus, surface sialic acids were specifically required for the periodate-induced stimulation of DNA synthesis in calf lym- phocytes.

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Surface Alterations in Oxidized Lymphocytes 1869

TABLE VII

Effect of neuraminidase pretreatment of lymphocytes on DNA synthesis

Calf lymphocytes, 12.5 x lO%nl were incubated with Vibrio cholera neuraminidase, 25 units/ml, for 90 min at 37” and then washed with Medium 199. A portion was then oxidized with NaIO,. Untreated, XaIO,-oxidized, neuraminidase-treated, or neuraminidase-treated oxi- dized cells were cultured at a density of 2 x lo* cells/ml for 48 hours, and DNA synthesis was measured. The results are means of five ex- periments.

Treatment of lymphocytes

Oxidation P. oulgaris

Neuraminidase E-PHA

Sti~n;laaion

(32 pglculture)

+ 11.9 + 18.9 - + 0.71 + + - 0.47

+ + 9.6

- - 1.0

DISCUSSION

Stimulation of DNA synthesis by NaIO, treatment of lymphocytes is of particular interest because (a) the events mediating lymphocyte transformation and initiation of DNA synthesis are not known, and their identification would have important implications in immunology and oncology; (b) one can study processes in NaIO,-transformed lymphocytes free of non-lymphoid proteins such as lectins or antigens which might coat the surface of responding lymphocytes; and (c) the stimulus for transformation, NaIO,, need be present for only a short period of time to commit responding cells to increased DNA synthesis (24).

Lectin binding to available glycoprotein receptor sites was used as a probe for structural alterations in the surface of oxidized lymphocytes. Induction of heterogeneity in the bind- ing sites, altered affinity of the lectin for the cell surface receptors, or changes in the number of receptors for the lectin each would reflect a structural change. The lectins studied were chosen because of their different glycopeptide specifici- ties. P. uulgaris E-PHA binds to glycopeptides with N-glycosidically linked oligosaccharides containing galactose - N-acetylglucosamine residues, especially if mannose is present in the “core” of the oligosaccharide chain (25). Mushroom PHA-B binds to glycopeptides with O-glycosidi- tally linked galactose - N-acetylgalactosamine residues (7). Con A binds to glycopeptides containing mannose in an (Y configuration.

Because each of the lectins used is a multivalent molecule, it is likely that the numbers of binding sites calculated from the binding studies underestimates the actual number of receptor sites.

In limited studies of lectin binding at 4”, the amount of mushroom PHA-B bound to lymphocytes at 4” was only 37% of that at 25”, and the amount of P. uulgaris E-PHA bound at 4” was only 76% of that at 25”. Thus, lectin binding at 25”, used in all experiments reported under “Results,” is probably greater than if similar experiments were performed at O-4”, tempera- tures below the temperature-dependent phase transition pro- posed to occur in eukaryotic cell membranes (26). The higher temperature was chosen so that the study might also reflect conformational changes in lectin receptors occurring only in

more fluid plasma membranes. Binding was not studied after NaBH, reduction of otherwise untreated lymphocytes since NaBH, reduction alone did not alter the rate of DNA synthesis.

Although the cell suspensions studied consisted nearly entirely of small and medium lymphocytes, subpopulations of lymphocytes (T cells, B cells, or others) were not evaluated. Scatchard analyses and double reciprocal plots of the binding data both indicated only one class of receptor for each lectin. This suggests that receptors on various subpopulations of cells were identical, although the possibility remains that different lymphocyte subpopulations had different numbers of recep- tors. It is unlikely that leakage of any intracellular molecules modified the lectin binding to intact cells in these experiments, since all cells were washed in medium prior to the binding assay, since nearly all cells were viable, and since the binding assays were performed immediately after oxidation or oxida- tion and reduction.

Results of the binding studies documented a significant increase in numbers of P. uulgaris and mushroom PHA-B receptor sites on oxidized lymphocytes. The oxidized receptors appeared to be homogeneous by Scatchard analyses suggesting that the additional receptor sites were glycoproteins with oligosaccharides similar in structure to those of unoxidized lymphocytes. This is compatible with a rearrangement in surface molecules permitting additional lectin to bind to glycoprotein receptors of identical or similar structure, and does not imply production of new receptor sites. New synthesis of additional receptor sites is also unlikely, because of the brief duration of NaIO, exposure prior to performing the binding study.

It is of interest that the apparent affinity of P. oulgaris E-PHA for nonoxidized and oxidized lymphocyte receptors was equal. This is consistent with observations on P. uulgaris E-PHA receptors of human erythrocyte glycopeptides, which bind P. uulgaris E-PHA equally well before and after neurami- nidase treatment (7). In contrast, the apparent affinity of mushroom PHA-B for oxidized calf lymphocyte receptors was increased compared to receptors on untreated cells. This conformational change in oxidized lymphocyte receptors may be related to oxidation of membrane sialic acids, since mush- room PHA-B has been shown to.have a much greater affinity for erythrocyte glycopeptides after removal of sialic acid (7).

Con A binding to lymphocytes was not significantly changed by oxidation, or oxidation plus reduction, indicating that the changes in mushroom PHA-B and P. uulgaris E-PHA binding were related to specific membrane alterations. Possible expla- nations for the differences are: (a) Con A binds to a surface structure distinct from P. uulgaris E-PHA and mushroom PHA-B on calf lymphocytes; (b) undetected surface conforma- tional changes are insufficient to permit more Con A molecules to bind; and (c) maximal amounts of Con A are bound even to unoxidized lymphocytes. Further evidence that alterations of receptor sites by oxidation are specific is that NaBH,-reduc- tion of lymphocytes reduced the number of binding sites for both P. uulgaris E-PHA and mushroom PHA-B to levels equal to or below those of untreated cells, but did not change the binding characteristics of Con A.

Analysis of the composition of oxidized plasma membranes revealed only a change in sialic acids. This suggests that only mild Malapradian oxidation occurred under the conditions of periodate used in these experiments, and that more severe Malapradian oxidation of hexoses and hexosamines within the oligosaccharide chains of glycoproteins or glycolipids did not

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1870 Surface Alterations in Oxidized Lymphocytes

occur. However, since analyses were performed on crude membrane preparations containing many different glyco- proteins and glycolipids, the techniques used might not detect oxidation of hexose, hexosamine, or amino acid residues accounting for less than 10% of the total. In addition, these experiments did not study whether structural changes occurred in glycolipid fractions as well. However, it is unlikely that oxidation of glycolipids would result in altered binding of the lectins studied, since purified mammalian cell membrane receptors for these lectins are glycoproteins (7, 25, 27). The lack of major composition changes in oligosaccharide content of the oxidized plasma membranes in this study suggests, therefore, that the altered lectin binding reflects a conformational change in glycoproteins due to oxidation of sialic acids.

The nature of the oxidation of sialic acids was incompletely studied in the experiments reported. Although there is good evidence for complete conversion of N-acetylneuraminic acid to NeuNAc-7-ald by periodate in the calf lymphocytes (as in sialoglycopeptides and erythrocytes (21, 28, 29)), the result of N-glycolylneuraminic acid oxidation is not characterized, since the oxidized products of N-glycolylneuraminic acid co- chromatographed with unaltered N-glycolylneuraminic acid. Studies are currently in progress to identify the oxidation products of N-glycolylneuraminic acid and of calf lymphocyte membranes by gas chromatography. This is important since 93% of the incorporation of NaB3H, was into N-glycolyl- neuraminic acid derivatives and since N-glycolylneuraminic acid accounts for 53% (30) to 63% (31) of bovine fibrinogen or bovine serum glycoproteins.

Acknowledgments-Dr. 0. 0. Blumenfeld (Albert Einstein College of Medicine, Bronx, N. Y.) generously provided a sample of NeuNAc-7 for comparison. The Star Meat Packing Co. kindly donated the calf submandibular lymph nodes. The assistance of Ms. Anneke Zimmerman, Ms. Marjorie Ash, and Ms. Mildred Rosenberg in preparing this manuscript is appre- ciated.

1 Parker, J. W., O’Brien, R. L., Lukes, R. J., and Steiner, J. (1972) Lancet 1,103-104

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Neuraminidase treatment of lymphocytes prevented trans- formation by subsequent periodate oxidation. This suggests that certain sialic acid-containing plasma membrane mole- cules are necessary for NaIO,-induced transformation of calf lymphocytes. Intracellular oxidation by NaIO,, although it occurs,* is not capable of initiating and sustaining lymphocyte transformation without the presence of surface sialic acid.

Lowry, 0. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-2i5

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It has been observed in other cell systems that cell-cell interaction is necessary for, or plays a major role in lymphocyte transformations (32, 33). However, in calf lymph node lympho- cytes, neither direct cell-cell interaction nor soluble mediators are necessary for maximal lymphocyte transformation.3 These studies did not identify which lymphocyte subpopulation was transformed by oxidation.

Kornfeld. R., and Siemers. C. (1974) J. Biol. Chem. 249, 1295-1:~01 Earl, D. C. h’., and Korner. A. (1965) Biochem. J. 94, ~‘LI-xJ Liao, T.-H., Gallop, P. M., and Blumenfeld, 0. 0. (1973) J. Biol.

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A mechanism proposed for the mediation of growth, DNA synthesis, or morphologic transformation of cells, is that a change in plasma membrane conformation or composition in some way signals the control process within the cell to change from a “resting” state to a “stimulated” or transformed state. This hypothesis has been applied to fibroblast growth in tissue culture (34, 35), to transformation of lymphocytes by lectins and anti-immunoglobulin antibody, and to stimulation of DNA synthesis in lymphocytes by oxidation (3, 36). These data support this hypothesis. Experiments are in progress to iden- tify the sialic acid-containing membrane glycoproteins whose oxidation is associated with lymphocyte transformation.

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REFERENCES

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C A Presant and S ParkerSurface alterations in calf lymphocytes oxidized by sodium periodate.

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