Immunochemistry,1975.Vol. 12,pp. 9-17. PergamonPress. Prinledin Great Britain
ANTIGENIC A N D I M M U N O G E N I C PROPERTIES OF MEMBRANE PROTEINS SOLUBILIZED BY SODIUM DESOXYCHOLATE, PAPAIN DIGESTION OR HIGH IONIC STRENGTH M. PRAT, G. T A R O N E and P. M. C O M O G L I O * Department of Human Anatomy, University of Torino, School of Medicine, 10126 Torino, Italy
(Received 11 July 1974) Abstract--Comparison between antigens solubilized from MOPC-460 mouse plasmacytoma cell membranes by means of three methods (treatment with a surfactant: sodium desoxycholate (DOC); high ionic strength, using 3 M potassium chloride (3 M KCI); insolubilized papain (PAP) digestion) is reported. DOC displayed the greatest quantitative efficiency and brought more than 80 per cent of the membrane proteins in solution, as well as about 15 per cent of the protein-bound sugars. 3 M KC1 and papain solubilized 16 per cent and 10 per cent proteins and 3 per cent and 5 per cent sugars respectively. SDS-acrylamide electrophoresis showed that DOC also solubilized the greatest number of proteins and demonstrated the disadvantages associated with PAP digestion. In the rabbit, however, DOC-solubilized proteins displayed poor immunogenicity. In particular, precipitating antibodies against membrane antigens that were not equally solubilized by 3 M KC1 could not be obtained. It is deduced from what is known of surfactant mechanisms that DOC solubilizes both 'integral' and 'extrinsic' membrane proteins, whereas the ionic strength acts primarily on the latter. Cross-adsorption experiments with the antisera produced against antigens solubilized with the two agents showed that the great majority of the antibodies induced against antigens solubilized from cell membranes were directed against 'extrinsic' proteins.
INTRODUCTION
It has become clear that an understanding of many important functions of eukaryote cells can be obtained via a study of the structure of their plasma membrane, particularly that of the macromolecules exposed on its surface. Identification and purification of the latter requires their solubilization. Once the components of the membrane have been identified by isolation of the pure membrane (for review see: De Pierre and Karnovsky, 1973) or selective labeling of the outer surface of the cell (Maddy, 1964; Berg, 1969; Bretscher, 1971; Phillips and Morrison, 1971 ; Tarone et al., 1973; Comoglio et al., 1974), the use of polar solvents is needed in order to employ the most sensitive methods of analysis, particularly the immunochemical ones. Solubilization must, however, provide for maximum yield and minimum interference with structural characteristics of native membrane molecules. The various methods proposed have given rise to two basic strategies in the solubilization of membrane antigens (Kahan, 1973): dissociation of non-covalent forces and cleavage of covalent bonds. These methods have been designed for different purposes and applied to a variety of models. Their direct * Author to whom correspondence should be addressed: Istituto di Anatomia Umana Cso. M.D'Azeglio 52, 10126 Torino Italy.
comparison has not been attempted. Much attention has been directed to the specific study of histocompatibility antigens and there are no clear indications of the suitability of any given method in cases where the composition of plasma membrane proteins is to be studied. The present paper compares the chemical and immunological features of products solubilized from cell membranes using the three more efficient and commonly employed methods: surfactant treatment, high ionic strength, and proteolytic digestion. Each method solubilizes a number of proteins with common characteristics and a certain number whose immunological and chemical features are specific for the treatment employed. MATERIALS AND METHODS
Cells Cells used in these experiments were obtained from MOPC-460 plasmocytomas kindly supplied by Dr. H. Eisen and Dr. E. Simms (Washington Univ. St. Louis Mo. USA) and maintained by serial subcutaneous transplantations in syngeneic Balb/C mice. Tumour cells were stored frozen at -40°C until used. Preparation of membranes 'Crude' membrane fractions were obtained as follows. Every step was performed at 0°C to avoid proteolytic degradation. Ten grams of minced tissue were suspended in 100
10
M. PRAT, G. TARONE and P. M. COMOGLIO
ml NaC1 0-15 M, phosphate buffered 10 mM, pH 7.2 (PBS), NaN 3 1 mM. Cells were disrupted by Dounce homogenization or, for large scale preparations, by cavitation for 5 sec. The homogenate was diluted 1 : 1 with 60~o sucrose solution in 10 mM phosphate buffer. The homogenate was layered on a cushion of 45~ sucrose and centrifuged at 400 g for 30 min to eliminate nuclei and whole cells; supernatants were saved and interface bands were recentrifuged once at 800 g for 30 min on the same cushion. The first and second supernatants were combined and centrifuged at 34,000 g for 20 min; pellets were then resuspended in PBS-azide and spun at 10,000 g for 10 min. Sediments were discarded and supernatants were further centrifuged at 34,000 g for 20 min and washed with PBS-azide until optical density of the last supernatant at 280 mm reached a value of less than 0-100 O.D. units. Solubilization Three different agents were studied: (1) high ionic strength, using 3 M potassium chloride solutions (3 M KCI), according to Reisfeld e t al. (1971). Each gram (wet wt) of membranes was suspended in 10 ml 3 M KCI, 10 mM phosphate buffered at pH 7.2 and vigorously stirred overnight at + 4°C. (2) a surfactant, sodium desoxycholate (DOC), by treating pellets with a 2~o (w/v) solution in 0.05 M barbital buffer, pH 8-2. One gram of membranes was suspended in 25 ml of DOC solution and stirred overnight at + 4°C. (3) enzymatic digestion, using insoluble papain. Papain was insolubilized by coupling to carboxymethyl-cellulose (Michael and Ewens, 1949). One gram of membranes was suspended in 0-15 M NaC1, 0.01 M Tris--HC1 buffer, pH 8.0, and incubated with 1 mg insoluble papain (PAP) activated with 1 ml 0.5 M cystein and 0'2 ml 50 mM EDTA. The final volume was brought to 10 ml and suspensions were incubated at + 37°C for 15 min. Samples solubilized by each of the three methods were centrifuged at 105,000 g for 90 min. Supernatants referred to as solubilized membrane antigens (SMA) were dialysed against Tris-HC1 buffer, pH 8.0. Removal of 3 M KC1 from membrane antigens led to the formation of a viscous material derived from the solution. This material, which was only partially solubi|ized by DNAse digestion was easily removed by centrifugation.
All the biochemical comparisons described in this paper were based on the study of several batches of solubilized material. Acrylamide gel electrophoresis The sodium dodecyl sulphate disc-gel electrophoresis method of Maizel (1971) was followed. SMA and 'crude' membrane samples, in triplicate, containing 150-200 /tg proteins, were heated at 100°C for 2 min in 100/tl 6 mM Tris-phosphoric acid buffer, pH 6-7, 1!?/oosodium dodecyl sulfate, 1~ mercaptoethanok 8°J~, sucrose and I~,o bromophenol blue as tracking dye. 9~o acrylamide gels, with a 3 ~ upper spacer, were polymerized in 0"1~o sodium dodecyl sulfate: runs were performed in 50 mM Tris-glycine buffer, pH 8-5, 0'1~o sodium dodecyl sulfate. Gels were fixed, stained and scanned in a Joice Loebl (Chromoscan) densitometer. Calibration was achieved by simultaneous separate runs of the following standards: cytochrome C (mol. wt = 12,000~ concanavalin A (mol. wt = 27,000), ovoalbumin (mol. wt = 43,000l bovine serum albumin (mol. wt = 67,000) and lactoperoxidase (mol. wt = 78,000). Production of antisera Rabbit antisera against solubilized membrane antigens were prepared by immunizing animals with three monthly spaced subcutaneous injections. Each SMA preparation was injected in a group of six New Zealand pedigreed rabbits. The number of proteins determined on the basis of the number obtained in disc-gel electrophoresis was used to estimate the antigen dose required. An average of 500 #g protein for each band was injected in each animal, emulsified 1 : 1 in Freund's complete adjuvant. Precipitin reactions Titration of precipitating antibodies, produced by rabbits immunized with membrane antigens solubilized by the three different agents, was performed by the micromethod described by Maurer (1971), with minor modifications: samples were not heated at + 37°C to avoid proteolytic digestion of SMAs, and immunoprecipitates were quantified by the Lowry method. Immune precipitations in gels were carried out, in accordance with Ouchterlony's double diffusion technique, in 1°/0agarose in 0.05 M barbital buffer, pH 8.4, l mM azide.
Analytical procedures Protein concentrations were determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. 'Crude' membrane protein concentrations were determined after complete solubilization in 10~o sodium dodecyl sulfate (SDS) at 100°C for 3 min. Lipids were extracted three times with chloroform-methanol according to the method of Weinstein et al. (1969), and their concentration was determined by the method of Chiang et al. (1957). Carbohydrates were measured by the method of Dische (1955), using glucose as standard, after removal of DOC by extensive dialysis. Interference due to the contamination of nucleic acid pentoses was minimized by heating samples at 100°C for 2 min to inactivate endogenous proteolytic enzymes and by digesting with purified DNAse and RNAse ( 10 #g/ml) at + 37°C for 1 hr. After TCA precipitation, samples were dialysed against distilled water. DNA and RNA were determined by the diphenylamine method (Burton, 1956) and the orcinol reaction (Dische, 1955a) respectively.
Cross adsorption of immune sera In cross-adsorption experiments, immune sera from each rabbit group were adsorbed through columns of Sepharose 4B (Pharmacia) to which SMAs obtained by the two other solubilization procedures were covalently bound by cyanogen bromide technique (Ax6n et al., 1967). Alternatively, sera were absorbed in incubation with appropriate aliquots of SMAs, followed by removal of the precipitate by centrifugation. RESULTS Chemical analysis o f S M A s The m e m b r a n e fraction prepared under the conditions described in the m e t h o d section was composed of 90~0 (w/w) water. The electron microscope picture of the final pellet showed that m e m b r a n e s were isolated in the form of sealed vesicles full of buffer solution. Dried vesicles were composed of a b o u t 78~o (w/w) pro-
11
Immunochemical Properties of Solubilized Membrane Antigens Table 1. Non dialysable-moleculessolubilized from 1 g of crude membranes Solubilizing agent
Proteins mg
~o
mg
~o
2~o DOC 3 M KC1 PAPAIN
62.8 +_ 0.8 12.6 _ 4.2 10.9 _+ 2.5
82.6 _+ 0.9 16.5 +_ 2.2 14.3 + 3.0
67.7 _+ 0.1 0.38 _ 0.03 0.80 +_ 0-03
> 100 1.6 3.4
TCA-precipitable carbohydrates ug Vo
Lipids
teins, 24 per cent lipids and about 2 per cent carbohydrates. DNA and RNA, as contaminants, were also found at about 0'1 per cent and 0.2 per cent respectively. As can be seen in Table 1, the three solubilizing agents yielded differently composed products. DOC proved the most effective protein solubilizing agent: 82 per cent of the total membrane proteins were recovered in DOC-SMA preparations. After treatment with 3 M KC1, only 16 per cent of the proteins were recovered while papain digestion released 14 per cent. DOC was also the most effective solubilizing agent for protein-bound carbohydrates: 13 per cent of total TCA-precipitable membrane sugars were recovered in the DOC-SMA preparation, whereas KC1 treatment and papain digestion released only 3 per cent and 4 per cent. The ratio /~g carbohydrates solubilized/mg proteins, however, was almost two times higher in papainSMA (9.1) than in DOC-SMA (4'7) and KCI-SMA (5"0), showing that proteolytic digestion preferentially solubilized proteins with carbohydrate moieties. However, these data underestimate the amount of total sugars released by papain, since enzymatic digestion broke off small glycopeptides that were lost after dialysis and TCA precipitation. As can be seen in Table 1, more lipids were found in DOC-SMA than the total measured in the 'crude' membrane fractions. This apparently paradoxical result is explained by the fact that in aqueous buffers bile salts (which are also measured by the assay) bind to the hydrophobic portions of membrane proteins. Based on molar ratio 414/80,000 (mol. wt of DOC/an estimate average of membrane protein mol. wt), we calculated that a protein molecule of 8 x 104 daltons binds about 200 DOC molecules. After dialysis against buffers containing 0.1~ DOC, from 1 to 3 per cent lipids were found in SMAs solubilized with KC1 or papain digestion, though as much as 30 per cent was found in both before dialysis. Since (as discussed below) direct action of the salt or the enzyme on lipids is very unlikely, this result is attributed to the release of phospholipids in micellar form as a secondary consequence of the removal of proteins from membrane. A significant estimate of how much contaminant was brought into solution by each agent was calculated from the amount of nucleic acids solubilized from the 'crude' membrane preparation. DOC solubilized almost 100 per cent of both DNA and RNA trapped in, or bound to, the membrane vesicles, while KCI released 60 per cent and 15 per cent respectively. Very
300 ___40 64_+3 100 +_ 4
13.0 2.7 4.3
low values were obtained with papain (less than 15 per cent).
SDS-Electrophoretic analysis Proteins solubilized by DOC, 3 M KCI or papain digestion were separated by SDS-9~o acrylamide disc gel electrophoresis and patterns in different runs were compared with those given by the whole membrane completely dissolved by SDS-mercaptoethanol at 100°C. The latter showed 45 incompletely resolved bands, corresponding to the same number of proteins or protein classes, spread over a large mol. wt range (Fig. 1). In DOC-SMA samples 31 bands of proteins of different molecular weight were seen (Fig. 2). KC1-SMA showed 21 bands (Fig. 3) and papain-SMA 16 (Fig. 4). Each band was numbered according to its calculated relative mobility (Rm) to establish how many and which bands were common to the three SMAs. As can be seen in Table 2, complete Rm identity was shown only by seven components four of which (protein 1, 2, 3 and 6) had a mol. wt of less 25,000 daltons, two (protein 16 and 17) close to 50,000 daltons and one (protein 24) 90,000 daltons. DOC-SMA and KC1-SMA showed 16 bands with identical mobility, DOC-SMA and papain-SMA nine
E
t~ t~
®
2"s
4"o 6o M.W, x 10 a
eb ~bo
®
Fig. l. Electrophoretic profile of proteins from membrane preparations completely dissolved by treatment with l ~ o SDS, l~o fl-mercaptoethanol at 100°C for 2 min. Gels were stained with Coomassie blue and scanned for light absorption at 620 nm. The mol. wt calibration was based on simultaneous separate runs of the standards listed under Materials and Methods. The top of the gel is on the right.
12
M. PRAT, G. TARONE and P. M. COMOGLIO o
I 0
e
DOC-SMA
14 19i i22 24 : 171 1 8 : : !! :23 8 : 6ii910 '3~il.6!.i ii'i !25 ~! i z 1.2j
o ~.
i
~o
oO.
!!
'
1,5 1B 3.4
E E O
26!
i i
ri
KC|-~
'i
:
3,
291
0
6 o io
25
4() M,W, X
60
8'0
1()0
*
e
a's
103
Fig. 2. Electrophoretic profile of DOC-solubilized membrane proteins in SDS-9% acrylamide gels: Thirty-one bands of different mol. wt were separated and numbered progressively according to their relative mobility. The same numbers are used for the visible bands in Figs. 4-6. Comparison can thus he made between all the patterns. The top of the gel is on the right. and papain-SMA and KCI-SMA seven (Figs. 2-4 and Table 2). Five bands in KCI-SMA had not corresponding bands in either D O C - S M A pattern or papain-SMA pattern. Similarly, seven bands in the papain-SMA were lacking in the other two SMAs. Two bands (protein 5 and 25) were present in D O C - S M A and p a p a i n SMA, but not in KCI-SMA. No bands were common to KC1-SMA and papain-SMA only. These results were reproducible using different batches of each SMA and were obtained with freshly prepared SMAs. Storage at + 4°C produced alterations in the pattern. The intensity and number of higher mol. wt bands were lowered, while new low mol. wt bands appeared. This was attributed to proteolytic degradation and was particularly strong in KC1-SMA (Fig. 5).
,b
i2,7
2|
6b eb 1oo
®
M.W. x 103
Fig. 3. Electrophoretic profile of membrane proteins solubilized by high ionic strength (3 M KC1). Bands with numbers greater than 31 have no equivalents in the patterns obtained with DOC and papain digestion. The top of the gel is on the right.
PAP- SM A
o m
E
b 2.5
o o
39! 411! ::!
[
Immunological properties of SMAs The amount of precipitating antibodies induced by the three SMAs was measured by precipiting reaction in tubes. With the exception of two rabbits, individual responses obtained within each group showed minimal variations. Each serum gave against its owp immunogen a rather regular curve. Equivalence was reached in a narrow concentration of antigen, 50-100 #g (protein) by all three systems. As can be seen in Fig. 6, the curve for anti-KCI-SMA antisera and its im-
®
2~
4o
eo
so ~00
®
M.W. x 103 Fig. 4. Electrophoretic profile of membrane proteins solubilized by papain digestion for 15 min at + 37°C. Bands with numbers greater than 36 have no equivalents in the patterns obtained with DOC and KCI. The top of the gel is on the right.
Table 2. Relative electrophoretic mobility of m e m b r a n e proteins solubilized by different agents ~' 50 ~ Migration
0
" Each band is identified by a n u m b e r based in its relative electrophoretic mobility. Circled n u m b e r s indicate protein bands which were c o m m o n to more than one pattern.
2~oDOC @@@ 4 @@@ 8 @ '0 @ @ 13 14 @ @ @ @ 19 20 21 @ 23 @ @ 26 @ 28 @ 30 31 3MKCl @ ~ 32 @@ @ 33 @ @ @@@@ 34 @ @ 35 @ 36 @ PAPAIN @@@ @@ 37 @ @ 38 39 40 41 @@ 42 43
Agent
I~l 100
U.
,=
o"
e-~
e~
o
~,
o
14
M. PRAT, G. TARONE and P. M. COMOGLIO --1
100
8O
I:I, •~
ec
o.
ol (D
40
ci
3.3
2o
10
2'5
4b M,W.
ab
sb 16o
®
x 10 3
Fig. 5. Electrophoretic profile of membrane proteins solubilized by 3 M KC1 after protracted storage at +4°C. Comparison with Fig. 4 shows that there are fewer proteins with a mol. wt 50,000 and that their peaks are lower. Unnumbered peaks indicate bands not found in Fig. 4. These changes appear to be the result of endogenous KCI-SMA proteolysis. The top of the gel is on the right.
munogen was three and five times higher than that for a n t i - P A P - S M A and a n t i - D O C - S M A , respectively, against their correspondent immunogens. SMAs prepared with DOC, KC1 or papain were precipitated in agar or agarose against their corresponding rabbit antisera and against those raised against SMA solubilized by the other two methods. KC1-SMA tested against its corresponding antiserum gave four thick bands, three of them fused, with lines of complete identity with those given by the a n t i - D O C - S M A antiserum, and two with those given by the anti-papainSMA antiserum (Table 3). A similar pattern was obtained with D O C - S M A . Papain-SMA showed only two bands common to the pattern developed by its corresponding anti-papain-SMA antiserum and those developed by anti-KCI-SMA and a n t i - D O C - S M A (Table 3). Testing each antiserum with different SMAs gave similar results. Different solubilization procedures may lead to the appearance of membrane antigens with specific immunochemical properties. To investigate this hypothesis, each antiserum was adsorbed by each of the two non-corresponding SMAs and retested by immunoprecipitation as above. Both internal absorption and adsorption via chromatography through insoluble
I
I
I
i
~)
50
100
200 pg S M A added
300
4 0
Fig. 6. Quantitative precipitin curves with membrane antigens solubilized by different agents and their corresponding rabbit antisera. (©): reaction between KCI-SMA and anti-KC1-SMA antiserum;(&): reaction between PAPSMA and anti-PAP-SMA antiserum; (e): reaction between DOC SMA and anti-DOC-SMA antiserum. Each curve was obtained by reacting 0"05 ml of antiserum with increasing amounts of SMA, as shown on the abscissae.
antigen columns were performed. The latter method enables each antiserum to react with a large excess of antigens. This avoids false results due to incomplete adsorption owing to the possible low concentration of some antigen. Results of cross-adsorption are summarized in Table 3. Anti-papain-SMA antisera were completely adsorbed by both KC1-SMA and D O C - S M A .
Table 3. Cross-precipitation and cross-adsorbtion patterns between membrane antigens solubilized by different agents and their corresponding antisera Rabbit antiserum a DOC a-KC1 a--PAP a-DOC a-KCI a-PAP a-DOC a KCI a-PAP
Unadsorb.
Adsorbed with DOC KC1 PAP
A. Tested against KCI-SMA 3" 4 0 2 0 B. Tested against DOC SMA 3 0 3 0 2 0 C. Tested against PAP-SMA 2 0 2 0 2 0
0 0 0
1 1 0
0 0
1 2 0
0 0 0
0 0
" Number of visible bands scored after staining.
Immunochemical Properties of Solubilized Membrane Antigens
1oo
80 0.
L
"e
'~ 60 Q.
._= ~ 4O eh
20
lo 5b
1~o
2~o
36o
4~o
pg S M A added
Fig. 7. Quantitative precipitin curve with membrane antigens solubilized by DOC and antiserum raised against membrane antigens solubilized by 3 M KCI (e . . . . e). Curves obtained with reaction between KC1-SMA and an anti-KCI-SMA antiserum (@ O), and DOC-SMA and an anti-DOC-SMA antiserum (,e) are shown for comparison. Anti-DOC-SMA antisera were completely adsorbed by KC1-SMA; when adsorbed by PAP-SMA, they still reacted with both DOC-SMA and KC1-SMA to give a single band. Similarly, adsorption of anti-KCI-SMA antisera with DOC-SMA completely removed any precipitating antibody. However, adsorption with PAP-SMA left one precipiting band when antisera were tested against KC1-SMA and (unexpectedly) two bands when tested against DOC-SMA. This shows that anti-KC1-SMA antisera contain antibodies that react with an antigen or group of antigens in D O C SMA that is not recognized by the corresponding antiDOC-SMA antisera. One only explanation is possible: these antigens are present in both DOC-SMA and KC1-SMA, since DOC-SMA was able to adsorb anti-KCI-SMA antisera completely. They are only able to elicit precipitating antibodies in KC1-SMA, however. This hypothesis was checked by a quantitative reaction with anti-KC1-SMA antisera and increasing amounts of DOC-SMA. As expected, DOC-SMA yielded five times more precipitate with the anti-KC1SMA antisera than with its corresponding antisera. The curve obtained reached the same value as that in the system obtained by testing KC1-SMA and antiKC1-SMA antisera (Fig. 7). DISCUSSION Among the solubilizing agents we studied, desoxycholate proved the most efficient one in quantitative
15
terms since solubilized more than 80 per cent of the proteins in entire membrane. This was due to its almost complete dissolution of the membrane by interfering with the stabilizing hydrophobic interactions. By associating with the lipophile portion of proteins it keeps them in solution by interacting with water molecules. Even 'integral' proteins (Singer and Nicolson, 1972) are dissolved in this way. Interaction between DOC and the lipophile portion of such proteins is relatively stable (Helenius and Simons, 1972) and leaves them with a net negative charge due to the carboxyl groups of the DOC molecule (Tzagaloff and Penefsky, 1971) and hence particular immunochemical properties (see later). Treatment with 3 M KCI solubilizes fewer proteins. The increased ionic strength is effective on non-covalent forces only: theoretically at least, this means that only 'extrinsic' membrane proteins (Singer and Nicolson, 1972) are directly affected, these being bound to the core by means of the forces mentioned. As we shall see later, however, the KC1-SMA displayed distinct endogenous proteolytic activity, so that one may not exclude that a part of the solubilized proteins were derived from 'integral' proteins embedded in the lipid matrix. It is not clear why KCI also releases a certain fraction of lipids that are not bound to proteins. Extrinsic proteins solubilized by KC1 may stabilize the membrane structure: their removal would mean structural rearrangement of the membrane represented by the release of micellar lipid formations. Papain, on the other hand, solubilizes proteins only by indiscriminate cleavage of peptide bonds on the membrane surfaces. In other words, it preferentially solubilizes protein molecules protruding from these surfaces into the aqueous medium. This portion includes most of the oligosaccharides bound to the glycoproteins (Nicholson, 1974), so that a very high sugar: protein ratio is observed in PAP-SMA. During solubilization, however, the enzyme not only attacks the peptide bonds linking the part of the protein anchored to the lipid matrix to that free in the aqueous medium, but also acts within the polypeptide chains and destroys them in situ, or after solubilization. It is for this reason that papain gives the lowest yield of the three methods. PAP-SMA contains bands not found in DOC-SMA, attributable solely to the fragmentation of certain proteins. Examination of the peaks obtained on densitometric scansion of the gels shows a marked predominance of low-weight peptides over the larger proteins. KC1-SMA also offers bands not seen in the DOC-SMA pattern. It is clear from the mechanism of action of this salt that there can be no question of its selective solubilization of some component unaffected by DOC. This unexpected finding must thus be seen as the result of alteration of proteins common to the two SMAs, as result of endogenic proteolysis. On the immunological side, the greater quantity of proteins solubilized by DOC is not matched by a correspondingly greater number of precipitating antibodies in serum-producing animals. The precipitation
16
M. PRAT, G. TARONE and P. M. COMOGLIO
curves indicate an antibody response five times lower than that induced by antigens solubilized with KCI. Furthermore, as shown by the cross-adsorption experiments, antisera against DOC-SMA did not contain precipitating antibodies against any component that was not present in KCI-SMA, and the same was true of PAP-SMA. In general terms, therefore, treatment with DOC and with KC1, though not with papain, solubilize the same number of antigens from the membrane. This, however, runs counter to the electrophoretic patterns, which showed that DOC solubilizes at least nine proteins not solubilized by KC1. It must be assumed that the latter are not able to induce precipitating antibodies, at least detectable by the methods used. It must be kept in mind, in fact, that both assays used can detect only antibodies to antigens with two or more functional antigenic determinants per molecule, i.e. those capable of precipitating. Moreover, since rabbits were immunized with heterogeneous material, it is hard to exclude a priori that antigenic competition may have played some role in the lack of antibody response against some membrane proteins solubilized by DOC. However, an easier explanation for this may be sought in the presence of DOC molecules associated with these proteins in a stable manner. The immunogenic properties of a given molecule are, in fact, a function of its amino acid composition (Gill and Doty, 1961 ; Sela et al., 1962; Fuchs and Sela, 1963; Gill et al., 1967), charge (Sela and Fuchs, 1963; Maurer et al., 1966; Gill et al., 1967), shape (Sela et al., 1963; Gill et al., 1968), size (Gill, 1972), and solubility (Borek, 1972). DOC bound to amino acid residues could have a negative influence on immunogenicity by modifying protein shape and solubility. A more significant effect, however, would result from their imparting of a high negative charge and/or their preferential masking of amino acid radicals totally or partly hydrophobic. 'Integral' molecules associate with the greatest number of DOC molecules on account of their particular richness in non-polar amino acids. It may be supposed that it is these DOC-SMA proteins that fail to induce the production of precipitating antibodies. This view is supported by the observation that KCI-SMA, which do not solubilize all the 'integral' proteins, completely absorbs the antisera induced by DOC-SMA. Moreover, cross-adsorption tests and the crossed precipitation curves for DOC- and KC1-SMA and their antisera indicate that DOC-SMA contains molecules that are not able to induce precipitating antibodies, but capable of specifically binding the precipitating antibodies induced by the corresponding proteins when solubilized with KC1. The conclusion may be drawn that DOC molecules associated with membrane proteins may in some cases interfere only with their ability to induce a response in vivo even though they do not interfere with the in vitro binding of antigenic determinants and the corresponding antibodies. This is in line with a series of observations showing that a portion of the macromolecule
other than the antigenic determinant is involved in interaction with the immunocompetent cell (Rajewsky and Rottlander, 1967; Grenn et al., 1968; Byrt and Ada, 1969; Parish and Ada, 1969; Rajewsky et al., 1969; Spliter et al., 1970; Senyk et al., 1971) and that the antigenic determinant appears to involve a smaller area of the molecule than the portion required for interaction with the immune competent cell (Gill, 1972). Acknowledgemen~The authors are indebted with Dr. G. Filogamo for encouragement and support. The skillful technical assistance of Miss M. R. Amedeo and Mrs. M. Lagna is gratefully acknowledged.This work was supported by the Italian National Research Council (C.N.R.).
REFERENCES
Ax~n R., Porath J. and Ernback S. (1967) Nature, Lond. 214, 1302. Berg H. (1969) Biochim. biophys. Acta 183, 65. Bretscher M. (1971) J. molec. Biol. 58, 775. Borek F. (1972) lmmunogenicity (Edited by Borek F.), p. 45. North-Holland, Amsterdam. Burton K. (1956) Biochem. J. 62, 315. Byrt C. R. and Ada G. (1969) Immunology 17, 153. Chiang S. F., Gessert C. F. and Lowry D. H. (1957) Curt. List reed. Lit. 33, 113. Comoglio P. M., Fuhrer J. P., Buck C. A. and Warren L. (1974) Biochemistry (submitted). De Pierre J. W. and Karnovsky M. L. (1973) J. cell. Biol. 56, 275. Dische Z. (1955) Methods of Biochemical Analysis (Edited by Glick D.), Vol. 2, p. 313. Interscience, New York. Dische Z. (1955a) The Nucleic Acids (Edited by Chargaff E. and Davidson J. N.), Vol. 1, p. 285. Academic Press, New York. Fuchs S. and Sela M. (1963) Biochem. J. 87, 70. Gill T. J. and Doty P. (1961) J. Biochem. 236, 2677. Gill T. J., Kunz H. W. and Papermaster D. S. (1967) J. biol. Chem. 242, 3308. Gill T. J., Papermaster D. S., Kunz H. W. and Marfey P. S. (1968) J. biol. Chem. 243, 287. Gill T. J. (1972) Immunogenicity (Edited by Borek F.), p. 5. North-Holland, Amsterdam. Green J., Paul W. and Benacerraf B. (1968) J. exp. Med. 127, 43. Helenius A. and Simons K. (1972) J. biol. Chem. 247, 3656. Kahan B. D. (1973) Methods in Cancer Research 9, 283. Lowry D. H., Rosebrough N. J, Farr A. L. and Randall R. T. (1951) J. biol. Chem. 193, 265. Maddy A. H. (1964) Biochim. biophys. Acta 88, 390. Maizel J. V. (1971) Methods in Virology (Edited by Maramorosch K. and Koprowski H.), Vol. 5, p. 315. Academic Press, New York. Maurer P. H., Gerulat B. F. and Pinchuck P. (1966) J. Immun. 97, 306. Maurer P. H. (1971) Methods in Immunology and Immunochemistry (Edited by Williams C. A. and Chase M. W.), Vol. 3, p. 41. Academic Press, New York. Michael F. and Ewens J. (1949) Macromol. Chem. 3, 200. Nicolson G. L. (1974) Int. Rev. Cytol. (in press). Parish C. R. and'Ada G. (1969) Immunology 17, 153. Phillips D. and Morrison M. (1971) Biochemistry 10, 1766. Rajewsky K. and Rottlander E. (1967) Cold Spring. Harb. Symp. quant. Biol. 22, 547.
Immunochemical Properties of Solubilized Membrane Antigens Rajewsky K., Schirrmacher V., Nase S. and Jerne N. (1969) J. exp. Med. 129, 1131. Reisfeld R. A., Pellegrino M. A. and Kahan D. B. (1971) Science 172, 1134. Sela M., Fuchs S. and Arnon R. (1962) Biochem. J. 85, 223. Sela M. and Fuchs S. (1963) Biochim. biophys. Acta 74, 796. Sela M., Fuchs S. and Feldman M. (1963) Science 139, 342. Senyk G., Niteiki D. and Goodman J. W. (1971) Science 171, 407. Singer S. G. and Nicolson G. L. (1972) Science 175, 720.
17
Spliter L., Benjamini E., Young D., Kaplan H. and Fudenberg H. (1970) J. exp. Med. 131, 133. Tarone G., Prat M. and Comoglio P. M. (1973) Biochim. biophys. Acta 311,214. Tzagaloff A. and Penefsky H. (197 l) Methods in Enzymolooy (Edited by Jakoby W. B.), Vol. 22, p. 219. Academic Press, New York. Weinstein D. H., Marsh J. B., Glick M. C. and Warren L. (1969) J. biol. Chem. 244, 4103.
ANTIGENIC A N D I M M U N O G E N I C PROPERTIES OF MEMBRANE PROTEINS SOLUBILIZED BY SODIUM DESOXYCHOLATE, PAPAIN DIGESTION OR HIGH IONIC STRENGTH M. PRAT, G. T A R O N E and P. M. C O M O G L I O * Department of Human Anatomy, University of Torino, School of Medicine, 10126 Torino, Italy
(Received 11 July 1974) Abstract--Comparison between antigens solubilized from MOPC-460 mouse plasmacytoma cell membranes by means of three methods (treatment with a surfactant: sodium desoxycholate (DOC); high ionic strength, using 3 M potassium chloride (3 M KCI); insolubilized papain (PAP) digestion) is reported. DOC displayed the greatest quantitative efficiency and brought more than 80 per cent of the membrane proteins in solution, as well as about 15 per cent of the protein-bound sugars. 3 M KC1 and papain solubilized 16 per cent and 10 per cent proteins and 3 per cent and 5 per cent sugars respectively. SDS-acrylamide electrophoresis showed that DOC also solubilized the greatest number of proteins and demonstrated the disadvantages associated with PAP digestion. In the rabbit, however, DOC-solubilized proteins displayed poor immunogenicity. In particular, precipitating antibodies against membrane antigens that were not equally solubilized by 3 M KC1 could not be obtained. It is deduced from what is known of surfactant mechanisms that DOC solubilizes both 'integral' and 'extrinsic' membrane proteins, whereas the ionic strength acts primarily on the latter. Cross-adsorption experiments with the antisera produced against antigens solubilized with the two agents showed that the great majority of the antibodies induced against antigens solubilized from cell membranes were directed against 'extrinsic' proteins.
INTRODUCTION
It has become clear that an understanding of many important functions of eukaryote cells can be obtained via a study of the structure of their plasma membrane, particularly that of the macromolecules exposed on its surface. Identification and purification of the latter requires their solubilization. Once the components of the membrane have been identified by isolation of the pure membrane (for review see: De Pierre and Karnovsky, 1973) or selective labeling of the outer surface of the cell (Maddy, 1964; Berg, 1969; Bretscher, 1971; Phillips and Morrison, 1971 ; Tarone et al., 1973; Comoglio et al., 1974), the use of polar solvents is needed in order to employ the most sensitive methods of analysis, particularly the immunochemical ones. Solubilization must, however, provide for maximum yield and minimum interference with structural characteristics of native membrane molecules. The various methods proposed have given rise to two basic strategies in the solubilization of membrane antigens (Kahan, 1973): dissociation of non-covalent forces and cleavage of covalent bonds. These methods have been designed for different purposes and applied to a variety of models. Their direct * Author to whom correspondence should be addressed: Istituto di Anatomia Umana Cso. M.D'Azeglio 52, 10126 Torino Italy.
comparison has not been attempted. Much attention has been directed to the specific study of histocompatibility antigens and there are no clear indications of the suitability of any given method in cases where the composition of plasma membrane proteins is to be studied. The present paper compares the chemical and immunological features of products solubilized from cell membranes using the three more efficient and commonly employed methods: surfactant treatment, high ionic strength, and proteolytic digestion. Each method solubilizes a number of proteins with common characteristics and a certain number whose immunological and chemical features are specific for the treatment employed. MATERIALS AND METHODS
Cells Cells used in these experiments were obtained from MOPC-460 plasmocytomas kindly supplied by Dr. H. Eisen and Dr. E. Simms (Washington Univ. St. Louis Mo. USA) and maintained by serial subcutaneous transplantations in syngeneic Balb/C mice. Tumour cells were stored frozen at -40°C until used. Preparation of membranes 'Crude' membrane fractions were obtained as follows. Every step was performed at 0°C to avoid proteolytic degradation. Ten grams of minced tissue were suspended in 100
10
M. PRAT, G. TARONE and P. M. COMOGLIO
ml NaC1 0-15 M, phosphate buffered 10 mM, pH 7.2 (PBS), NaN 3 1 mM. Cells were disrupted by Dounce homogenization or, for large scale preparations, by cavitation for 5 sec. The homogenate was diluted 1 : 1 with 60~o sucrose solution in 10 mM phosphate buffer. The homogenate was layered on a cushion of 45~ sucrose and centrifuged at 400 g for 30 min to eliminate nuclei and whole cells; supernatants were saved and interface bands were recentrifuged once at 800 g for 30 min on the same cushion. The first and second supernatants were combined and centrifuged at 34,000 g for 20 min; pellets were then resuspended in PBS-azide and spun at 10,000 g for 10 min. Sediments were discarded and supernatants were further centrifuged at 34,000 g for 20 min and washed with PBS-azide until optical density of the last supernatant at 280 mm reached a value of less than 0-100 O.D. units. Solubilization Three different agents were studied: (1) high ionic strength, using 3 M potassium chloride solutions (3 M KCI), according to Reisfeld e t al. (1971). Each gram (wet wt) of membranes was suspended in 10 ml 3 M KCI, 10 mM phosphate buffered at pH 7.2 and vigorously stirred overnight at + 4°C. (2) a surfactant, sodium desoxycholate (DOC), by treating pellets with a 2~o (w/v) solution in 0.05 M barbital buffer, pH 8-2. One gram of membranes was suspended in 25 ml of DOC solution and stirred overnight at + 4°C. (3) enzymatic digestion, using insoluble papain. Papain was insolubilized by coupling to carboxymethyl-cellulose (Michael and Ewens, 1949). One gram of membranes was suspended in 0-15 M NaC1, 0.01 M Tris--HC1 buffer, pH 8.0, and incubated with 1 mg insoluble papain (PAP) activated with 1 ml 0.5 M cystein and 0'2 ml 50 mM EDTA. The final volume was brought to 10 ml and suspensions were incubated at + 37°C for 15 min. Samples solubilized by each of the three methods were centrifuged at 105,000 g for 90 min. Supernatants referred to as solubilized membrane antigens (SMA) were dialysed against Tris-HC1 buffer, pH 8.0. Removal of 3 M KC1 from membrane antigens led to the formation of a viscous material derived from the solution. This material, which was only partially solubi|ized by DNAse digestion was easily removed by centrifugation.
All the biochemical comparisons described in this paper were based on the study of several batches of solubilized material. Acrylamide gel electrophoresis The sodium dodecyl sulphate disc-gel electrophoresis method of Maizel (1971) was followed. SMA and 'crude' membrane samples, in triplicate, containing 150-200 /tg proteins, were heated at 100°C for 2 min in 100/tl 6 mM Tris-phosphoric acid buffer, pH 6-7, 1!?/oosodium dodecyl sulfate, 1~ mercaptoethanok 8°J~, sucrose and I~,o bromophenol blue as tracking dye. 9~o acrylamide gels, with a 3 ~ upper spacer, were polymerized in 0"1~o sodium dodecyl sulfate: runs were performed in 50 mM Tris-glycine buffer, pH 8-5, 0'1~o sodium dodecyl sulfate. Gels were fixed, stained and scanned in a Joice Loebl (Chromoscan) densitometer. Calibration was achieved by simultaneous separate runs of the following standards: cytochrome C (mol. wt = 12,000~ concanavalin A (mol. wt = 27,000), ovoalbumin (mol. wt = 43,000l bovine serum albumin (mol. wt = 67,000) and lactoperoxidase (mol. wt = 78,000). Production of antisera Rabbit antisera against solubilized membrane antigens were prepared by immunizing animals with three monthly spaced subcutaneous injections. Each SMA preparation was injected in a group of six New Zealand pedigreed rabbits. The number of proteins determined on the basis of the number obtained in disc-gel electrophoresis was used to estimate the antigen dose required. An average of 500 #g protein for each band was injected in each animal, emulsified 1 : 1 in Freund's complete adjuvant. Precipitin reactions Titration of precipitating antibodies, produced by rabbits immunized with membrane antigens solubilized by the three different agents, was performed by the micromethod described by Maurer (1971), with minor modifications: samples were not heated at + 37°C to avoid proteolytic digestion of SMAs, and immunoprecipitates were quantified by the Lowry method. Immune precipitations in gels were carried out, in accordance with Ouchterlony's double diffusion technique, in 1°/0agarose in 0.05 M barbital buffer, pH 8.4, l mM azide.
Analytical procedures Protein concentrations were determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. 'Crude' membrane protein concentrations were determined after complete solubilization in 10~o sodium dodecyl sulfate (SDS) at 100°C for 3 min. Lipids were extracted three times with chloroform-methanol according to the method of Weinstein et al. (1969), and their concentration was determined by the method of Chiang et al. (1957). Carbohydrates were measured by the method of Dische (1955), using glucose as standard, after removal of DOC by extensive dialysis. Interference due to the contamination of nucleic acid pentoses was minimized by heating samples at 100°C for 2 min to inactivate endogenous proteolytic enzymes and by digesting with purified DNAse and RNAse ( 10 #g/ml) at + 37°C for 1 hr. After TCA precipitation, samples were dialysed against distilled water. DNA and RNA were determined by the diphenylamine method (Burton, 1956) and the orcinol reaction (Dische, 1955a) respectively.
Cross adsorption of immune sera In cross-adsorption experiments, immune sera from each rabbit group were adsorbed through columns of Sepharose 4B (Pharmacia) to which SMAs obtained by the two other solubilization procedures were covalently bound by cyanogen bromide technique (Ax6n et al., 1967). Alternatively, sera were absorbed in incubation with appropriate aliquots of SMAs, followed by removal of the precipitate by centrifugation. RESULTS Chemical analysis o f S M A s The m e m b r a n e fraction prepared under the conditions described in the m e t h o d section was composed of 90~0 (w/w) water. The electron microscope picture of the final pellet showed that m e m b r a n e s were isolated in the form of sealed vesicles full of buffer solution. Dried vesicles were composed of a b o u t 78~o (w/w) pro-
11
Immunochemical Properties of Solubilized Membrane Antigens Table 1. Non dialysable-moleculessolubilized from 1 g of crude membranes Solubilizing agent
Proteins mg
~o
mg
~o
2~o DOC 3 M KC1 PAPAIN
62.8 +_ 0.8 12.6 _ 4.2 10.9 _+ 2.5
82.6 _+ 0.9 16.5 +_ 2.2 14.3 + 3.0
67.7 _+ 0.1 0.38 _ 0.03 0.80 +_ 0-03
> 100 1.6 3.4
TCA-precipitable carbohydrates ug Vo
Lipids
teins, 24 per cent lipids and about 2 per cent carbohydrates. DNA and RNA, as contaminants, were also found at about 0'1 per cent and 0.2 per cent respectively. As can be seen in Table 1, the three solubilizing agents yielded differently composed products. DOC proved the most effective protein solubilizing agent: 82 per cent of the total membrane proteins were recovered in DOC-SMA preparations. After treatment with 3 M KC1, only 16 per cent of the proteins were recovered while papain digestion released 14 per cent. DOC was also the most effective solubilizing agent for protein-bound carbohydrates: 13 per cent of total TCA-precipitable membrane sugars were recovered in the DOC-SMA preparation, whereas KC1 treatment and papain digestion released only 3 per cent and 4 per cent. The ratio /~g carbohydrates solubilized/mg proteins, however, was almost two times higher in papainSMA (9.1) than in DOC-SMA (4'7) and KCI-SMA (5"0), showing that proteolytic digestion preferentially solubilized proteins with carbohydrate moieties. However, these data underestimate the amount of total sugars released by papain, since enzymatic digestion broke off small glycopeptides that were lost after dialysis and TCA precipitation. As can be seen in Table 1, more lipids were found in DOC-SMA than the total measured in the 'crude' membrane fractions. This apparently paradoxical result is explained by the fact that in aqueous buffers bile salts (which are also measured by the assay) bind to the hydrophobic portions of membrane proteins. Based on molar ratio 414/80,000 (mol. wt of DOC/an estimate average of membrane protein mol. wt), we calculated that a protein molecule of 8 x 104 daltons binds about 200 DOC molecules. After dialysis against buffers containing 0.1~ DOC, from 1 to 3 per cent lipids were found in SMAs solubilized with KC1 or papain digestion, though as much as 30 per cent was found in both before dialysis. Since (as discussed below) direct action of the salt or the enzyme on lipids is very unlikely, this result is attributed to the release of phospholipids in micellar form as a secondary consequence of the removal of proteins from membrane. A significant estimate of how much contaminant was brought into solution by each agent was calculated from the amount of nucleic acids solubilized from the 'crude' membrane preparation. DOC solubilized almost 100 per cent of both DNA and RNA trapped in, or bound to, the membrane vesicles, while KCI released 60 per cent and 15 per cent respectively. Very
300 ___40 64_+3 100 +_ 4
13.0 2.7 4.3
low values were obtained with papain (less than 15 per cent).
SDS-Electrophoretic analysis Proteins solubilized by DOC, 3 M KCI or papain digestion were separated by SDS-9~o acrylamide disc gel electrophoresis and patterns in different runs were compared with those given by the whole membrane completely dissolved by SDS-mercaptoethanol at 100°C. The latter showed 45 incompletely resolved bands, corresponding to the same number of proteins or protein classes, spread over a large mol. wt range (Fig. 1). In DOC-SMA samples 31 bands of proteins of different molecular weight were seen (Fig. 2). KC1-SMA showed 21 bands (Fig. 3) and papain-SMA 16 (Fig. 4). Each band was numbered according to its calculated relative mobility (Rm) to establish how many and which bands were common to the three SMAs. As can be seen in Table 2, complete Rm identity was shown only by seven components four of which (protein 1, 2, 3 and 6) had a mol. wt of less 25,000 daltons, two (protein 16 and 17) close to 50,000 daltons and one (protein 24) 90,000 daltons. DOC-SMA and KC1-SMA showed 16 bands with identical mobility, DOC-SMA and papain-SMA nine
E
t~ t~
®
2"s
4"o 6o M.W, x 10 a
eb ~bo
®
Fig. l. Electrophoretic profile of proteins from membrane preparations completely dissolved by treatment with l ~ o SDS, l~o fl-mercaptoethanol at 100°C for 2 min. Gels were stained with Coomassie blue and scanned for light absorption at 620 nm. The mol. wt calibration was based on simultaneous separate runs of the standards listed under Materials and Methods. The top of the gel is on the right.
12
M. PRAT, G. TARONE and P. M. COMOGLIO o
I 0
e
DOC-SMA
14 19i i22 24 : 171 1 8 : : !! :23 8 : 6ii910 '3~il.6!.i ii'i !25 ~! i z 1.2j
o ~.
i
~o
oO.
!!
'
1,5 1B 3.4
E E O
26!
i i
ri
KC|-~
'i
:
3,
291
0
6 o io
25
4() M,W, X
60
8'0
1()0
*
e
a's
103
Fig. 2. Electrophoretic profile of DOC-solubilized membrane proteins in SDS-9% acrylamide gels: Thirty-one bands of different mol. wt were separated and numbered progressively according to their relative mobility. The same numbers are used for the visible bands in Figs. 4-6. Comparison can thus he made between all the patterns. The top of the gel is on the right. and papain-SMA and KCI-SMA seven (Figs. 2-4 and Table 2). Five bands in KCI-SMA had not corresponding bands in either D O C - S M A pattern or papain-SMA pattern. Similarly, seven bands in the papain-SMA were lacking in the other two SMAs. Two bands (protein 5 and 25) were present in D O C - S M A and p a p a i n SMA, but not in KCI-SMA. No bands were common to KC1-SMA and papain-SMA only. These results were reproducible using different batches of each SMA and were obtained with freshly prepared SMAs. Storage at + 4°C produced alterations in the pattern. The intensity and number of higher mol. wt bands were lowered, while new low mol. wt bands appeared. This was attributed to proteolytic degradation and was particularly strong in KC1-SMA (Fig. 5).
,b
i2,7
2|
6b eb 1oo
®
M.W. x 103
Fig. 3. Electrophoretic profile of membrane proteins solubilized by high ionic strength (3 M KC1). Bands with numbers greater than 31 have no equivalents in the patterns obtained with DOC and papain digestion. The top of the gel is on the right.
PAP- SM A
o m
E
b 2.5
o o
39! 411! ::!
[
Immunological properties of SMAs The amount of precipitating antibodies induced by the three SMAs was measured by precipiting reaction in tubes. With the exception of two rabbits, individual responses obtained within each group showed minimal variations. Each serum gave against its owp immunogen a rather regular curve. Equivalence was reached in a narrow concentration of antigen, 50-100 #g (protein) by all three systems. As can be seen in Fig. 6, the curve for anti-KCI-SMA antisera and its im-
®
2~
4o
eo
so ~00
®
M.W. x 103 Fig. 4. Electrophoretic profile of membrane proteins solubilized by papain digestion for 15 min at + 37°C. Bands with numbers greater than 36 have no equivalents in the patterns obtained with DOC and KCI. The top of the gel is on the right.
Table 2. Relative electrophoretic mobility of m e m b r a n e proteins solubilized by different agents ~' 50 ~ Migration
0
" Each band is identified by a n u m b e r based in its relative electrophoretic mobility. Circled n u m b e r s indicate protein bands which were c o m m o n to more than one pattern.
2~oDOC @@@ 4 @@@ 8 @ '0 @ @ 13 14 @ @ @ @ 19 20 21 @ 23 @ @ 26 @ 28 @ 30 31 3MKCl @ ~ 32 @@ @ 33 @ @ @@@@ 34 @ @ 35 @ 36 @ PAPAIN @@@ @@ 37 @ @ 38 39 40 41 @@ 42 43
Agent
I~l 100
U.
,=
o"
e-~
e~
o
~,
o
14
M. PRAT, G. TARONE and P. M. COMOGLIO --1
100
8O
I:I, •~
ec
o.
ol (D
40
ci
3.3
2o
10
2'5
4b M,W.
ab
sb 16o
®
x 10 3
Fig. 5. Electrophoretic profile of membrane proteins solubilized by 3 M KC1 after protracted storage at +4°C. Comparison with Fig. 4 shows that there are fewer proteins with a mol. wt 50,000 and that their peaks are lower. Unnumbered peaks indicate bands not found in Fig. 4. These changes appear to be the result of endogenous KCI-SMA proteolysis. The top of the gel is on the right.
munogen was three and five times higher than that for a n t i - P A P - S M A and a n t i - D O C - S M A , respectively, against their correspondent immunogens. SMAs prepared with DOC, KC1 or papain were precipitated in agar or agarose against their corresponding rabbit antisera and against those raised against SMA solubilized by the other two methods. KC1-SMA tested against its corresponding antiserum gave four thick bands, three of them fused, with lines of complete identity with those given by the a n t i - D O C - S M A antiserum, and two with those given by the anti-papainSMA antiserum (Table 3). A similar pattern was obtained with D O C - S M A . Papain-SMA showed only two bands common to the pattern developed by its corresponding anti-papain-SMA antiserum and those developed by anti-KCI-SMA and a n t i - D O C - S M A (Table 3). Testing each antiserum with different SMAs gave similar results. Different solubilization procedures may lead to the appearance of membrane antigens with specific immunochemical properties. To investigate this hypothesis, each antiserum was adsorbed by each of the two non-corresponding SMAs and retested by immunoprecipitation as above. Both internal absorption and adsorption via chromatography through insoluble
I
I
I
i
~)
50
100
200 pg S M A added
300
4 0
Fig. 6. Quantitative precipitin curves with membrane antigens solubilized by different agents and their corresponding rabbit antisera. (©): reaction between KCI-SMA and anti-KC1-SMA antiserum;(&): reaction between PAPSMA and anti-PAP-SMA antiserum; (e): reaction between DOC SMA and anti-DOC-SMA antiserum. Each curve was obtained by reacting 0"05 ml of antiserum with increasing amounts of SMA, as shown on the abscissae.
antigen columns were performed. The latter method enables each antiserum to react with a large excess of antigens. This avoids false results due to incomplete adsorption owing to the possible low concentration of some antigen. Results of cross-adsorption are summarized in Table 3. Anti-papain-SMA antisera were completely adsorbed by both KC1-SMA and D O C - S M A .
Table 3. Cross-precipitation and cross-adsorbtion patterns between membrane antigens solubilized by different agents and their corresponding antisera Rabbit antiserum a DOC a-KC1 a--PAP a-DOC a-KCI a-PAP a-DOC a KCI a-PAP
Unadsorb.
Adsorbed with DOC KC1 PAP
A. Tested against KCI-SMA 3" 4 0 2 0 B. Tested against DOC SMA 3 0 3 0 2 0 C. Tested against PAP-SMA 2 0 2 0 2 0
0 0 0
1 1 0
0 0
1 2 0
0 0 0
0 0
" Number of visible bands scored after staining.
Immunochemical Properties of Solubilized Membrane Antigens
1oo
80 0.
L
"e
'~ 60 Q.
._= ~ 4O eh
20
lo 5b
1~o
2~o
36o
4~o
pg S M A added
Fig. 7. Quantitative precipitin curve with membrane antigens solubilized by DOC and antiserum raised against membrane antigens solubilized by 3 M KCI (e . . . . e). Curves obtained with reaction between KC1-SMA and an anti-KCI-SMA antiserum (@ O), and DOC-SMA and an anti-DOC-SMA antiserum (,e) are shown for comparison. Anti-DOC-SMA antisera were completely adsorbed by KC1-SMA; when adsorbed by PAP-SMA, they still reacted with both DOC-SMA and KC1-SMA to give a single band. Similarly, adsorption of anti-KCI-SMA antisera with DOC-SMA completely removed any precipitating antibody. However, adsorption with PAP-SMA left one precipiting band when antisera were tested against KC1-SMA and (unexpectedly) two bands when tested against DOC-SMA. This shows that anti-KC1-SMA antisera contain antibodies that react with an antigen or group of antigens in D O C SMA that is not recognized by the corresponding antiDOC-SMA antisera. One only explanation is possible: these antigens are present in both DOC-SMA and KC1-SMA, since DOC-SMA was able to adsorb anti-KCI-SMA antisera completely. They are only able to elicit precipitating antibodies in KC1-SMA, however. This hypothesis was checked by a quantitative reaction with anti-KC1-SMA antisera and increasing amounts of DOC-SMA. As expected, DOC-SMA yielded five times more precipitate with the anti-KC1SMA antisera than with its corresponding antisera. The curve obtained reached the same value as that in the system obtained by testing KC1-SMA and antiKC1-SMA antisera (Fig. 7). DISCUSSION Among the solubilizing agents we studied, desoxycholate proved the most efficient one in quantitative
15
terms since solubilized more than 80 per cent of the proteins in entire membrane. This was due to its almost complete dissolution of the membrane by interfering with the stabilizing hydrophobic interactions. By associating with the lipophile portion of proteins it keeps them in solution by interacting with water molecules. Even 'integral' proteins (Singer and Nicolson, 1972) are dissolved in this way. Interaction between DOC and the lipophile portion of such proteins is relatively stable (Helenius and Simons, 1972) and leaves them with a net negative charge due to the carboxyl groups of the DOC molecule (Tzagaloff and Penefsky, 1971) and hence particular immunochemical properties (see later). Treatment with 3 M KCI solubilizes fewer proteins. The increased ionic strength is effective on non-covalent forces only: theoretically at least, this means that only 'extrinsic' membrane proteins (Singer and Nicolson, 1972) are directly affected, these being bound to the core by means of the forces mentioned. As we shall see later, however, the KC1-SMA displayed distinct endogenous proteolytic activity, so that one may not exclude that a part of the solubilized proteins were derived from 'integral' proteins embedded in the lipid matrix. It is not clear why KCI also releases a certain fraction of lipids that are not bound to proteins. Extrinsic proteins solubilized by KC1 may stabilize the membrane structure: their removal would mean structural rearrangement of the membrane represented by the release of micellar lipid formations. Papain, on the other hand, solubilizes proteins only by indiscriminate cleavage of peptide bonds on the membrane surfaces. In other words, it preferentially solubilizes protein molecules protruding from these surfaces into the aqueous medium. This portion includes most of the oligosaccharides bound to the glycoproteins (Nicholson, 1974), so that a very high sugar: protein ratio is observed in PAP-SMA. During solubilization, however, the enzyme not only attacks the peptide bonds linking the part of the protein anchored to the lipid matrix to that free in the aqueous medium, but also acts within the polypeptide chains and destroys them in situ, or after solubilization. It is for this reason that papain gives the lowest yield of the three methods. PAP-SMA contains bands not found in DOC-SMA, attributable solely to the fragmentation of certain proteins. Examination of the peaks obtained on densitometric scansion of the gels shows a marked predominance of low-weight peptides over the larger proteins. KC1-SMA also offers bands not seen in the DOC-SMA pattern. It is clear from the mechanism of action of this salt that there can be no question of its selective solubilization of some component unaffected by DOC. This unexpected finding must thus be seen as the result of alteration of proteins common to the two SMAs, as result of endogenic proteolysis. On the immunological side, the greater quantity of proteins solubilized by DOC is not matched by a correspondingly greater number of precipitating antibodies in serum-producing animals. The precipitation
16
M. PRAT, G. TARONE and P. M. COMOGLIO
curves indicate an antibody response five times lower than that induced by antigens solubilized with KCI. Furthermore, as shown by the cross-adsorption experiments, antisera against DOC-SMA did not contain precipitating antibodies against any component that was not present in KCI-SMA, and the same was true of PAP-SMA. In general terms, therefore, treatment with DOC and with KC1, though not with papain, solubilize the same number of antigens from the membrane. This, however, runs counter to the electrophoretic patterns, which showed that DOC solubilizes at least nine proteins not solubilized by KC1. It must be assumed that the latter are not able to induce precipitating antibodies, at least detectable by the methods used. It must be kept in mind, in fact, that both assays used can detect only antibodies to antigens with two or more functional antigenic determinants per molecule, i.e. those capable of precipitating. Moreover, since rabbits were immunized with heterogeneous material, it is hard to exclude a priori that antigenic competition may have played some role in the lack of antibody response against some membrane proteins solubilized by DOC. However, an easier explanation for this may be sought in the presence of DOC molecules associated with these proteins in a stable manner. The immunogenic properties of a given molecule are, in fact, a function of its amino acid composition (Gill and Doty, 1961 ; Sela et al., 1962; Fuchs and Sela, 1963; Gill et al., 1967), charge (Sela and Fuchs, 1963; Maurer et al., 1966; Gill et al., 1967), shape (Sela et al., 1963; Gill et al., 1968), size (Gill, 1972), and solubility (Borek, 1972). DOC bound to amino acid residues could have a negative influence on immunogenicity by modifying protein shape and solubility. A more significant effect, however, would result from their imparting of a high negative charge and/or their preferential masking of amino acid radicals totally or partly hydrophobic. 'Integral' molecules associate with the greatest number of DOC molecules on account of their particular richness in non-polar amino acids. It may be supposed that it is these DOC-SMA proteins that fail to induce the production of precipitating antibodies. This view is supported by the observation that KCI-SMA, which do not solubilize all the 'integral' proteins, completely absorbs the antisera induced by DOC-SMA. Moreover, cross-adsorption tests and the crossed precipitation curves for DOC- and KC1-SMA and their antisera indicate that DOC-SMA contains molecules that are not able to induce precipitating antibodies, but capable of specifically binding the precipitating antibodies induced by the corresponding proteins when solubilized with KC1. The conclusion may be drawn that DOC molecules associated with membrane proteins may in some cases interfere only with their ability to induce a response in vivo even though they do not interfere with the in vitro binding of antigenic determinants and the corresponding antibodies. This is in line with a series of observations showing that a portion of the macromolecule
other than the antigenic determinant is involved in interaction with the immunocompetent cell (Rajewsky and Rottlander, 1967; Grenn et al., 1968; Byrt and Ada, 1969; Parish and Ada, 1969; Rajewsky et al., 1969; Spliter et al., 1970; Senyk et al., 1971) and that the antigenic determinant appears to involve a smaller area of the molecule than the portion required for interaction with the immune competent cell (Gill, 1972). Acknowledgemen~The authors are indebted with Dr. G. Filogamo for encouragement and support. The skillful technical assistance of Miss M. R. Amedeo and Mrs. M. Lagna is gratefully acknowledged.This work was supported by the Italian National Research Council (C.N.R.).
REFERENCES
Ax~n R., Porath J. and Ernback S. (1967) Nature, Lond. 214, 1302. Berg H. (1969) Biochim. biophys. Acta 183, 65. Bretscher M. (1971) J. molec. Biol. 58, 775. Borek F. (1972) lmmunogenicity (Edited by Borek F.), p. 45. North-Holland, Amsterdam. Burton K. (1956) Biochem. J. 62, 315. Byrt C. R. and Ada G. (1969) Immunology 17, 153. Chiang S. F., Gessert C. F. and Lowry D. H. (1957) Curt. List reed. Lit. 33, 113. Comoglio P. M., Fuhrer J. P., Buck C. A. and Warren L. (1974) Biochemistry (submitted). De Pierre J. W. and Karnovsky M. L. (1973) J. cell. Biol. 56, 275. Dische Z. (1955) Methods of Biochemical Analysis (Edited by Glick D.), Vol. 2, p. 313. Interscience, New York. Dische Z. (1955a) The Nucleic Acids (Edited by Chargaff E. and Davidson J. N.), Vol. 1, p. 285. Academic Press, New York. Fuchs S. and Sela M. (1963) Biochem. J. 87, 70. Gill T. J. and Doty P. (1961) J. Biochem. 236, 2677. Gill T. J., Kunz H. W. and Papermaster D. S. (1967) J. biol. Chem. 242, 3308. Gill T. J., Papermaster D. S., Kunz H. W. and Marfey P. S. (1968) J. biol. Chem. 243, 287. Gill T. J. (1972) Immunogenicity (Edited by Borek F.), p. 5. North-Holland, Amsterdam. Green J., Paul W. and Benacerraf B. (1968) J. exp. Med. 127, 43. Helenius A. and Simons K. (1972) J. biol. Chem. 247, 3656. Kahan B. D. (1973) Methods in Cancer Research 9, 283. Lowry D. H., Rosebrough N. J, Farr A. L. and Randall R. T. (1951) J. biol. Chem. 193, 265. Maddy A. H. (1964) Biochim. biophys. Acta 88, 390. Maizel J. V. (1971) Methods in Virology (Edited by Maramorosch K. and Koprowski H.), Vol. 5, p. 315. Academic Press, New York. Maurer P. H., Gerulat B. F. and Pinchuck P. (1966) J. Immun. 97, 306. Maurer P. H. (1971) Methods in Immunology and Immunochemistry (Edited by Williams C. A. and Chase M. W.), Vol. 3, p. 41. Academic Press, New York. Michael F. and Ewens J. (1949) Macromol. Chem. 3, 200. Nicolson G. L. (1974) Int. Rev. Cytol. (in press). Parish C. R. and'Ada G. (1969) Immunology 17, 153. Phillips D. and Morrison M. (1971) Biochemistry 10, 1766. Rajewsky K. and Rottlander E. (1967) Cold Spring. Harb. Symp. quant. Biol. 22, 547.
Immunochemical Properties of Solubilized Membrane Antigens Rajewsky K., Schirrmacher V., Nase S. and Jerne N. (1969) J. exp. Med. 129, 1131. Reisfeld R. A., Pellegrino M. A. and Kahan D. B. (1971) Science 172, 1134. Sela M., Fuchs S. and Arnon R. (1962) Biochem. J. 85, 223. Sela M. and Fuchs S. (1963) Biochim. biophys. Acta 74, 796. Sela M., Fuchs S. and Feldman M. (1963) Science 139, 342. Senyk G., Niteiki D. and Goodman J. W. (1971) Science 171, 407. Singer S. G. and Nicolson G. L. (1972) Science 175, 720.
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Spliter L., Benjamini E., Young D., Kaplan H. and Fudenberg H. (1970) J. exp. Med. 131, 133. Tarone G., Prat M. and Comoglio P. M. (1973) Biochim. biophys. Acta 311,214. Tzagaloff A. and Penefsky H. (197 l) Methods in Enzymolooy (Edited by Jakoby W. B.), Vol. 22, p. 219. Academic Press, New York. Weinstein D. H., Marsh J. B., Glick M. C. and Warren L. (1969) J. biol. Chem. 244, 4103.
