Isolation and partial characterization of plasma membrane glycoproteins from normal and transformed mammalian cells employing plant lectin affinity chromatography

Experimental

ISOLATION

AND

PLASMA

Cell Research 109 (1977) 95-103

PARTIAL

MEMBRANE

NORMAL

AND

CELLS

GLYCOPROTEINS

TRANSFORMED

EMPLOYING

AFFINITY

CHARACTERIZATION

OF FROM

MAMMALIAN

PLANT

LECTIN

CHROMATOGRAPHY E. PEARLSTEIN

lrvingtor~

House

Institute.

Depurtmrnt ofPathology, Neil, York Near, York, N.Y. 10016. USA

University

Medicul

Center.

SUMMARY Lectins covalently bound to Sepharose 4B were employed, in the presence of 1% sodium deoxycholate, to purify glycoproteins from isolated plasma membranes of normal and transformed fibroblasts. Lectins used in this study were isolated from Lens culinaris (LcH) and Ricinus communis (RCA,) and were specific for o-glucose and o-mannose (LcH) and o-galactose (RCA,), respectively. Approx. 5 % of the total plasma membrane protein applied to the LcH-containing column bound and was eluted specifically, whereas approx. 3 % was bound and specifically eluted from the RCAr column. A comparison of bound glycoproteins from normal and transformed cells by SDS polyacrylamide gel electrophoresis demonstrated that a glycoprotein of 85000 D was present in substantially greater amounts in normal cell isolates. Another glycoprotein of 130000 D was found in higher concentration in transformed cell plasma membranes. The 85 000 D protein was shown to be a subunit of a serum protein of greater than 300000 D which has a specific affinity for the normal but not transformed cell surface. These results are discussed in relation to the differential agglutinability of normal and transformed cells by plant lectins.

Lectins with affinity for specific carbohydrate groups have been used to study the role of plasma membrane glycoproteins and glycolipids in the altered behavior of cells following transformation. It is now generally accepted that transformed cells are more readily agglutinable by low levels of lectins than are their normal cell counterparts [l]. However, normal cells are also agglutinable if (a) the concentration of lectin employed is sufficiently high [2]; (b) the cells are first treated with low levels of trypsin [3]; or (c) if they are tested during the mitotic phase of the cell cycle [4, 51. 7-771803

Despite the relative ease of agglutination of transformed cells by lectins compared with normal cells, it is known in most cases that both cell types bind equivalent amounts of radioactively labelled lectin [6, 71. It therefore appears that the differential agglutinability is not solely a function of total lectin binding, but may be a result of differences in the receptors for the lectins. The fluid mosaic model for plasma membrane structure [8] predicts that the increased agglutinability of transformed cells is due to an increase in the fluidity of their plasma membrane, which allows the lectinE.rl,

Cell

Rrs

109 (lY77)

96

Pearlstein

binding sites more freedom to achieve the tran 500-polyethylene glycol two phase polymer as described by Brunette & Till [14]. To meproper orientation and positioning for ag- system tabolically label cellular proteins, cells were cultured glutination. However, differences in the ag- for 24-48 h in E4 containing l/l0 concentration of L-leucine plus 10% dialysed serum supplemented with glutinability of cells might occur if qualita5 &i/ml of [3H-L]leucine (New England Nuclear, tively different receptors were expressed on 30-50 Cilmmole). The cells were harvested as described above except the 24 h incubation in serum-free the cell surface following transformation. medium was omitted. Lectin affinity chromatography of plasma membrane preparations in the presence of Affinity chromatography neutral detergents or bile salts [9] has been Purified plant lectins were covalently attached to CNBr-activated Sepharose 4B [1 1] at a concentration shown to be an effective method for puriof 1 mg protein/ml Sepharose 4B. Isolated plasma fying carbohydrate-containing components membranes were dissolved in 1% sodium deoxycholate (DOC, obtained from Sigma) spun at 100000 g of these membranes. The method has previfor 1 h, and the supematant applied to the Sepharoseously been employed to isolate glycoprolectin column previously equilibrated in 1% DOC. The column was washed with 1% DOC until the optical teins of eukaryotic cell plasma membranes density (OD,,,) of eluted material had returned to [IO] and viral coat proteins [Ill. background levels. Specifically bound material was in a sharp peak by application of a specifically In the present study this technique has eluted competitive monosaccharide (0.3 M a-methyl-obeen employed to compare plasma memmannoside in 1% DOC for LcH or 0.3 M D-galactoside in 1% DOC for RCA,). The column was re-usable with brane glycoproteins of baby hamster kidney little loss of binding activity, following extensive (BHK) cells and a polyoma virus trans- washing with 1% DOC. For prolonged storage, the columnsulfate, was equilibrated with 75% saturated ammoformed derivative, PyBHK. A serum corn- nium ponent of molecular weight greater than Afftnity chromatography of serum was performed in same fashion except that the serum was diluted 300000 D is specifically associated with the the 1 : 1 with PBS before application, and chromatography normal cell plasma membrane. A prelimiwas done in the presence of PBS instead of 1% DOC. nary report of this work has been pubPreparation of samplesfor gel lished [12]. electrophoresis [ 151 MATERIALS

AND

METHODS

Cells Low passage baby hamster kidney cells BHK2l/Cl3 (BHK), and a line of BHK cells transformed with polvoma virus (PvBHK) were grown in Dulbecco’s modification of ‘Eagle’s’ medium (E4) supplemented with 10 % calf serum. Cells were cultured at 37°C in a 5 % CO, atmosphere.

Lectins Plant lectins were isolated from Lens culinaris [ 111 and communus [13] by published procedures. The lectin from Lens culinaris (LcH) binds specifically to o-mannose and u-glucose. The Ricinus communis lectin (RCAr) has a specificity for o-galactose.

Ricks

Plasma membrane isolation To minimize serum protein contamination, cells were grown to confluence, washed twice with serum-free medium and placed into serum-free medium 24 h prior to harvesting. The cells were then harvested by scraping and the plasma membrane purified using the dexExp Cd Res 109 (1977)

The protein fractions were dialysed against 1% DOC to remove monosaccharide inhibitor, frozen, and lyophilized. Absolute ethanol was added to the dry residue, and after standing at -20’C for approx. 24 h, the precipitated protein was pelleted by centrifugation at 10000 a for 15 min. The supematant, containing DOC, wasdiscarded, and 10% TCA (OY) was added to the precipitate. After standing at 0°C for 30 min, the mixture was centrifuged at 10000 g for 15 min, and the supematant containing TCA was discarded. The pellet was again washed with cold ethanol, and after discarding the final supernatant, the protein pellet was dried under vacuum.

Polyacrylamide gel electrophoresis Dried protein was dissolved in sample buffer containing 2 % sodium dodecyl sulfate (SDS), 0.1 M Tris-HCl (pH 6.8) and 10% glycerol. Prior to electrophoresis, the samples were boiled for 2 min. When reduction of disultide bonds was desired, the dissolved samples were adjusted to 0.1 M dithiothreitol before boiling for 2 min. Polyacrylamide gel electrophoresis (PAGE) [ 16, 171 was performed in a slab gel [18] at the acrylamide monomer concentration indicated in the figure captions. After electrophoresis, the gels were stained with

Isolation

1, 5

10

15

20

30

Fig. I. Affinity chromatography of BHK plasma mem, brane proteins on an RCA,-Sepharose 4B column Chromatography was performed in 1% DOC.

Coomassie Brilliant Blue (0.5 a Coomassie. 520 ml H,O, 473 ml methanol, and ‘57 ml glacial acetic acid) to visualize protein bands. Proteins labelled with PH-Llleucine were visualized by fluorography (Kodak xray film, RP/R-54) after impregnating the gel with 25 diphenyloxazole (PPO) [ 191.

Immunodiffusion and immunoelectrophoresis Rabbit anti-bovine serum was kindly supplied by Dr N. J. Calvanico and standard immunodiffusion [20] techniques in 1% ion agar were employed. Immunodiffusion analysis of isolated plasma membranes and whole cell lysates were performed in gels containing 1% ion agar and 1% NP-40 dissolved in PBS.

RESULTS The elution profile of BHK plasma membrane proteins chromatographed on an RCA1 affinity column is shown in fig. 1. Ten ml of soluble plasma membrane proteins isolated from approx. 5~ IO9 cells was applied to the column at a flow rate of 0.5 ml/ min. The column was washed at 0.5 ml/min with 1% DOC until the ODzso had returned

of plasma

membrane

glycoproteins

97

to background. The specifically bound glycoproteins were eluted with a 0.3 M solution of competitive monosaccharide in 1% DOC (in this case 0.3 M n-galactose) at 1 ml/min. Affinity chromatography of similar samples on LcH columns yielded essentially identical patterns. Polyacrylamide gel electrophoresis of BHK and PyBHK plasma membrane proteins chromatographed on RCA1 and LcH affinity columns is shown in fig. 2. No significant differences can be seen in the Coomassie Blue staining patterns of unbound membrane proteins from normal BHK cells (LcH, 2b and RCA1, 2c) and transformed PyBHK cells (LcH, 2d and RCAi, 2~). On the other hand, comparison of the unbound and bound fractions show that a distinct subset of proteins (presumably glycoproteins) were bound to and specifically eluted from the lectin (cf fig. 2f-i vs b-e). It is also apparent that a more complex mixture of proteins is bound by LcH lectin than by RCA, lectin. This is shown in a comparison of plasma membrane proteins from BHK and PyBHK cells bound to LcH lectin (fig. 2f, i) with identical preparations of plasma membrane proteins bound to RCA, lectin (fig. 2g, h). Since the binding specificities of the lectins are known, these results must reflect differences in the amount of glycoproteins which bear the appropriate carbohydrate side chains in a configuration which is accessible for binding to the lectin. Selective binding of glycoproteins by the lectin columns was further demonstrated by rechromatography of the bound and specifically eluted proteins on the same lectin column. In such experiments greater than 90 % of applied glycoprotein was again bound to and specifically eluted from the column. In contrast, when the original plasma membrane preparation was applied to the lectin columns, only approx. 5% of the total Exp Cdl

Kes

109 C/977)

98

Pearlstein

abcdef

-2

Fig. 2. Coomassie-stained gel after SDS-polyacrylamide gel electrophoresis of plasma membrane proteins from normal or transformed cells fractionated on lectin affmity columns. Monomer concentration is 7.5 % and all samples have been reduced with 0.1 M DTT. (a) Marker-proteins: phosphorylase A (100000 D). BSA (68 000 D), and lactate dehydrogenase (36 000

D). Kev for remaining samples is cell tvpe. lectin. interacilon with lectm (bound or unbound); (b) BHK, LcH, unbound; (c) BHK, RCA,, unbound; (d) PyBHK, RCA,, unbound; (e) PyBHK, LcH, unbound; (f, BHK, LcH, bound; (g) BHK, RCA,, bound; (h) PyBHK, RCAt, bound; (i) PyBHK, LcH, bound; (i) same standards as in (u).

protein bound to the LcH column, and 3 % to the RCA1 column. Further comparison of the bound fractions from normal and transformed cells shows that although most glycoproteins are relatively unchanged following transformation, two differences are apparent. These differences are most easily visualized in those fractions bound to and specifically eluted from the RCA1 lectin. A glycoprotein of approx. 85000 D is present in plasma membrane preparations from normal BHK

cells (2 g, arrow I), but is greatly reduced, or absent, in preparations from transformed PyBHK cells (2h). Another glycoprotein of approx. 130000 D appears to be more prominent in preparations from the transformed PyBHK cells (fig. 2h, arrow 2) than in the normal BHK cells (fig. 2g). To differentiate between cellular proteins and contaminating serum proteins, cells were grown for 2448 h in the presence of [3H]leucine, plasma membranes prepared as before, and chromatographed on the

E.\-/I Cc//

Rr.\

/t/Y (IY77)

Isolation

a

of plasma

a

b

membrane

b

glycoproteins

99

c

Fig. 4. Coomassie-stained gel after SDS-polyacrylamide gel electrophoresis of serum proteins bound to RCA, affinity columns. Monomer concentration is 7.5% and samples were reduced with 0.1 M DTT. (a) Marker proteins: phosphorylase A (IO0000 D) and BSA (68000 D). Samples are the bound fraction from (h) calf serum and (c) human serum.

Fig. 3. Fluorograph after SDS-polyacrylamide gel electrophoresis of [3H]leucine-labelled plasma membrane glycoproteins bound to RCA, affinity columns. Monomer concentration is 7.5% and samples have been reduced with 0. I M DTT. Samples are the bound fraction from (a) BHK; (b) PyBHK cells.

RCA1 affinity column. Fig. PAGE pattern of [3H]leucine ma membrane glycoproteins specifically eluted from RCA,

3 shows the labelled plasbound to and lectin. A gly-

coprotein of approx. 250000 D which can be readily labelled by lactoperoxidase catalysed iodination of the cell surface of BHK, but not PyBHK [21], is detectable in the [3H]leucine RCA, bound fraction from BHK (fig. 3a, arrow l), but not PyBHK (fig. 36). A glycoprotein of 130000 D, which probably corresponds to the 130000 D glycoprotein observed by Coomassie staining (fig. 2h, arrow 2) is seen in the RCA1 bound plasma membrane glycoproteins from r3H]leucine labelled PyBHK cells (fig. 3a). In&/, cc// /?r.\IOY(I9771

100

Pearlstein

b

a

c

0.6

b

0.4

Fig.

5. Abscissa: fraction no.; ordinate: OD. Coomassie-stained SDS-polyacrylamide gel of RCA,-bound calf serum proteins before and after fractionation by chromatography on Sephadex G-150. For the gel, the monomer concentration is 5% and the

samples were unreduced. Samples are (a) calf serum, RCA,, bound, and the first (b) and second (c) peaks off the Sephadex G-150 column. The column dimensions were 2.5~ 100 cm and the flow rate was 15 ml/h. Five ml fractions were collected.

spection of the region of the gel where an 85000 D molecular weight protein would migrate shows no major [3H]leucinelabelled component, and, furthermore, no significant differences in the amount of label in this region in the plasma membranes of BHK and PyBHK cells. These results are consistent with the hypothesis that the 85 000 D Coomassie stained glycoprotein is a serum-derived component, which binds to and is purified with normal BHK plasma membranes, but not with those of polyoma virus-transformed PyBHK. In order to more directly investigate the possibility that the 85000 D glycoprotein might be serum-derived, 10 ml of calf serum (diluted 1 : 1 with PBS) was chromatographed on the RCA, lectin column. PAGE analysis of bound material (fig. 4) demonstrated a major glycoprotein of 85 000 D

bound to and specifically eluted from RCA1 lectin (fig. 4b). In a parallel experiment, human serum chromatographed on an RCA1 affinity column also contained a glycoprotein with the same lectin affinity and molecular weight (fig. 4c, arrow). Chromatography of calf serum in 1% DOC, instead of PBS, as was done during plasma membrane fractionation, yielded identical results to those obtained without detergent (data not shown). PAGE analysis of the RCA1 bound calf serum glycoproteins under non-reducing conditions (fig. 5 a) showed two major components of approx. 150000 and 300000 D, as well as some aggregated protein at the interface of the stacking and running gels. These two components were separated by chromatography in PBS on Sephadex G-150 (fig. 5) and purity established by PAGE

Exp Cell

Res 109 (1977)

Isolation

a

b

c

of plasma membrane

glycoproteins

101

tiserum (fig. 7). The antiserum was placed in the central well and calf serum glycoproteins bound to RCAi, plasma membranes from BHK cells, and plasma membranes from PyBHK cells in adjacent wells. The results demonstrated a reaction of identity between one of the RCA,-bound serum components and protein found in the BHK plasma membrane fraction. PyBHK plasma membranes failed to show the crossreacting component. To insure that the presence of the immunologically cross-reacting component in BHK plasma membranes was not an artefact of the plasma membrane isolation procedure, confluent dishes of BHK and PyBHK were washed live times with PBS and the cells directly lysed in 1% NP-40. Cell lysates were placed in immunodiffusion plates (fig. 8) and allowed to react with rabbit anti-bovine serum antiserum. The BHK cell lysate gave a single line reaction of identity with the serum glycoprotein which bound to RCA, lectin and chromatographed

Fin. 6. Coomassie-stained SDS-nolvacrvlamide gel of-RCA,-bound calf serum components fractionat;d on Sephadex G-150 in PBS. The acrylamide monomer concentration is 7.5% and the samples were reduced with 0.1 M DTT. (a) Marker protein: phosphorylase a (100000 D); (b) calf serum, RCA,, bound, first peak off Sephadex G-150; (c) calf serum, RCA,, bound second peak off Sephadex G- 150.

(fig. 5b, c). When the same samples were analyzed by PAGE in the presence of reducing agent (fig. 6), the first peak off G-150 contained the 85000 D component (fig. 6b, arrow 1) and a smaller molecular weight subunit of 25 000 D (fig. 6 b, arrow 2). Immunological identity between a serum glycoprotein and a component present in BHK plasma membrane fractions was established using rabbit antibovine serum an-

Fig. 7. Immunodiffusion in 1% ion agar gels containing 1% NP-40 of (a) calf serum proteins bound to RCA,; (b) solubilized BHK plasma membrane components; (c) solubilized PyBHK plasma membrane components; (d) rabbit anti-calf serum antiserum.

102

Pearlstein

Fig. 8. Immunodiffusion in 1% ion agar gels containing 1% NP-40 of (a) Sephadex G-150 first peak of RCAI-bound calf serum components; (b) BHK whole cell lysate; (c) PyBHK whole cell lysate; (d) rabbit anti-calf serum antiserum.

with the first peak off Sephadex G-150. Again, the PyBHK cell lysates failed to show the cross-reacting component. DISCUSSION We have applied the technique of lectin affinity chromatography to the study of the glycoprotein composition of plasma membranes isolated from normal and transformed fibroblasts. Solubilization and chromatography of purified plasma membranes was performed in 1% DOC. Lectins with specificity for u-galactose (RCA,) and for o-glucose or n-mannose (LcH) were used for affinity chromatography. RCA, lectin specifically binds approx. 3 % of the total plasma membrane protein, while LcH lectin binds slightly more (approx. 5%). These low percentages were not a function of protein excess relative to available lectin since in some experiments when half the amount Exp

Cdl

Rrs

109 (1977)

of total protein was applied, the percent of protein bound did not vary. Thus, the amount of glycoprotein possessing the correct carbohydrate moiety in the proper position for interaction with each lectin represents a small percentage of the total plasma membrane protein. The identification of lectin-bound proteins as glycoproteins has been substantiated by specific labelling of galactose residues using [3H]sodium borohydride and galactose oxidase prior to affinity chromatography. Under the conditions greater than 90 % of the label is bound by RCA, and (J. Smart, personal eluted with D-gdaCtOSe communication). Polyacrylamide gel electrophoresis of bound and unbound plasma membrane components demonstrates the selectivity of lectin affinity chromatography (see fig. 2 b, g). The major components of the bound material clearly differ from those of the unbound material. Also, the PAGE pattern of the RCA,-bound glycoproteins shows about 15 major components, compared to more than 50 major proteins present in the unfractionated plasma membranes. Lectins with additional carbohydrate specificities are available [22], and sequential chromatography of complex mixtures of glycoproteins, such as those found in plasma membranes and serum, should prove a valuable aid in the fractionation of specific components. Although most protein and glycoprotein components of the plasma membranes appear to be relatively unchanged following transformation, some differences in the expression of glycoproteins are apparent. A serum-derived glycoprotein of 85 000 D appears to be bound to and purified with the plasma membranes of normal, but not transformed cells. A glycoprotein of identical molecular weight and lectin binding spe-

Isolation

&cities can be isolated (fig. 3) and appears to be a subunit of a serum glycoprotein of approx. 300000 D (fig. 5). This serumderived glycoprotein is immunologically identical with a component which is present in the plasma membranes isolated from normal cells, and in whole cell lysates from normal cells. The detection of the crossreacting component in normal (BHK) whole cell lysates, but not in transformed (PyBHK) whole cell lysates, indicates that the presence of this protein in plasma membrane fractions is not an artefact of the membrane isolation procedure, which might have fractionated the normal and transformed plasma membranes unequally. A cell-derived glycoprotein of approx. 130000 D is found in substantially greater amounts in the transformed as compared with the normal cell plasma membrane. It is the subject of a separate report. Although many investigators have shown that a consequence of in vitro cellular transformation is an increased agglutinability of the cells by plant lectins, there is no universally accepted mechanism to explain the phenomenon. A factor that may contribute to the increased susceptibility of certain transformed cell lines to lectin-induced agglutination would be qualitative changes in lectin receptors on the cell surface. BHK cells are less agglutinable than PyBHK cells and have now been shown to have easily detectable amounts of an 85000 D serumderived glycoprotein on their plasma membranes, which is not present in significant quantities on the plasma membranes of PyBHK cells. This glycoprotein is bound efficiently by RCA, and LcH lectins. The BHK cells also show increased amount of a cell-derived glycoprotein of approx. 130000 D following transformation by polyoma virus (PyBHK).

of plusma

memhrune

103

glycoproteins

It is known that transformed cells have altered requirements for as yet poorly defined macromolecular serum factors [23]. The detection of a serum component with affinity for normal cell plasma membranes may provide insight into one possible mechanism for these altered requirements. The isolation of this protein should facilitate investigation of its biological function. Supported

in part by USPHS

Grant

no. AM

01431.

REFERENCES 1. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418. 2. Noonan, K D &Burger, M M, J cell biol59 (1973) 134. 3. Burger, M M, Proc natl acad xi US 62 (1969) 994. 4. Shoham, J & Sachs, L. Proc natl acad sci US 69 ( 1972) 2479. 5. Noonan, K D & Burger, M M, J biol them 248 ( 1973) 4286. 6. Cline, M J & Livingston, DC, Nature new biol232 (1971) 155. 7. Ozanne, B & Sambrook. J, Nature new biol 232 (1971) 156. 8. Singer, S J & Nicolson, G L, Science 175 (1972) 720. 9. Crumpton, M J & Parkhouse, R M E, FEBS lett 22 (1972) 210. IO. Smart, J E & HOIX, N. Nature 261 (1976) 314. Il. Hayman, M J & &mpton, M J, B&hem biophys res commun 47 (1972) 923. 12. Smart, J E, Pearlstein, E & Waterfield, M J, Biothem sot trans 2 (1974) 28. 13. Nicolson, G L, Blaustein. J & Etzler, M E, Biochemistry 13 (1974) 196. 14. Brunette, D M & Till. J E, J membr biol 5 (1971) 215. 15. Pearlstein, E & Seaver, J. Biochim biophys acta 426(1976)589. 16. Laemmli, U K, Nature 227 (1970), 680. 17. Studier, F W, Science 176 (1972) 367. 18. Maizel. J, Methods in virology (ed K Maramorosch & H Koprowski) vol. 5, p. 179. Academic Press, New York (1971). 19. Laskey, F A & Mills, A D, Eur j biochem 56 (1975) 335. 20. Ouchterlony, 0, Nature 215 (1958) 149. 21. Pearlstein, E & Waterfield. M D. Biochim bionhvs acta 362 (1974) I. 22 Sharon, N & Lis, H, Science 177 (1972) 949. 23 Halley , R W, Nature 258 (I 975) 487. Received Accepted

February 7, 1977 March 29, 1977