Study on the surface polypeptides isolated from human lymphoblastoid cells by ionic shock

863

Biochimica et Biophysica Acta, 444 (1976) 863--874 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 28045 STUDY ON THE SURFACE POLYPEPTIDES ISOLATED FROM HUMAN LYMPHOBLASTOID CELLS BY IONIC SHOCK

E.H.M. LOa, MUN H. NGa, W.S, NOa, SYLVIA LUIa and H.C. HOb aDepartment of Microbiology, University of Hong gong, Pathology Building, Queen Mary Hopsital Compound, and blnstitUte of Radiology and Oncology, Queen Elisabeth Hospital, Kowloon (Hong Kong)

(Received April 6th, 1976) Summary Ionic shock treatment in the presence of 10% glycerol is an efficient and selective method for extracting cell surface components from Raji cells and effecting the solubilization of up to 22 polypeptides. The m~jority of these shock-released polypeptides are accessible to lactoperoxidase radioiodination. Sera f r o m rabbits immunized against t h e s e soluble extracts are reactive against Raji cell surface as indicated by indirect m e m b r a n e immunofluorescence and agglutination assays.

Introduction Sequential treatment Of w h o l e h u m a n lymphoblastoid (Raji) cells with buffers of different ionic concentrations under Conditions of minimal cell lysis resulted in t h e release of cellular components which appeared to be largely Of cell surface origin. Some of these restdting extracts were found toelicit significantly greater in vitro cell-mediated immune responses with the peripheral leukocytes of nasopharyngeal carcinoma patients than with those of control cancer patients. On t h e other hand, b o t h groups of subjects displayed similar in vitro cell-mediated immune responses t o the purified protein derivatives o f Mycobacterium tuberculosis. The different ceil-mediated i m m u n e responses shown by these groups of subjects are believed to reflect nasopharyngeal carcinoma-specific tumor immunity in vivo a n d t h e discriminatory factors in the extracts might therefore be identified with the t u m o r antigens (Ng 'et: al. [1] ). we report here the further study and isolation of these factors and identification of the polypeptide contents of these Raji cell e x t r a c t s by acrylamide gel electrophoresis. We have also studied cellular localization Of these polypeptides using enzymic radioiodination and hyperimmune rabbit s e r a against these extracts.

864 Materials and Methods

Cell culture. Raji cells were propagated as described previously [ 1]. Lactoperoxidase radioiodination. This method is based on that described by Cone and Marchalonis [2]. Raji cells having a cellular viability of greater than 90% were used. They were washed twice and resuspended to give 2 • 107 cells per ml in Hank's buffer salt solution without phenol red. 20/~Ci Na125I (carrier free, Amersham~ England) was added per ml of cell suspension and horse radish lactoperoxidase (Calbiochem, U.S.) was added to give a final concentration of 1 • 10 -7 M. The reaction was initiated by adding H202 to give a final concentration of 44 p_M and allowed to proceed at room temperature for 10 rain. The cells were then washed three times with minimal essential medium (Eagles, Grand Island Biological, U.S.) containing 0.1 mM of sodium iodide, followed by two washes with Hank's buffer. Cellular viability as indicated by trypan blue exclusion, remained unchanged at the end of these manipulations and the labelled Raji cells were used immediately for studies. Ionic shock treatment. Radioiodinated or unlabelled Raji cells were extracted sequentially with Tris • HC1 buffer (0.02 M Tris. HC1, pH 7.2, containing 10% glycerol) and low EDTA buffer (Tris • HC1, 0.02 M; NaCI, 0.2 M; EDTA, 10 raM; 10% glycerol, pH 7.2) as described previously [1]. The resulting extracts are referred to as TS and ES, respectively. In some instances the extracts were further concentrated approx. 5-fold by vacuum dialysis. Cell fractionation. 2--4.107 ionic shock-treated or untreated cells, suspended in 2 ml Hank's buffer were freeze-thawed four times, layered onto 5 ml 0.5 M sucrose solution made up with Hank's buffer, and centrifuged at 105 000 X g for 2 h at 4°C. The resulting supernatant and pellet, hereafter referred to as the cytoplasmic and membrane fractions, respectively, were stored at --20°C until use. Sodium dodecylsulfate-polyacrylamide gel electrophoresis. Electrophoresis was carried out in 7% acrylamide gel according to the method of Nachman et al. [3] for 10 h at a constant current of 3.5 mA per gel. The gels were pre-electrophoresed for 2 h before use and all samples were heated at 100°C for 20 min in 0.1 M phosphate buffer pH 7.2 containing 2% each of sodium dodecylsulfate and 2-mercaptoethanol. Approx. 200--300 /~g protein were loaded onto each gel. The gels were stained with 0.01% Amidoblack 10B in 7% acetic acid and destained in 7% acetic acid. The gels containing radioactivity were later sliced to about 1--2 mm slices for radioactivity counting. Protein determination. The method of Lowry et al. [4] was used. Radioactivity counting. Aliquot samples were prepared as 10% trichloroacetic acid precipitates and collected onto GF/C glassfiber filters for radioactivity counting. The stained gel slices were counted without further processing. A Panax counter (England) was used throughout and the counting efficiency was 11%.

Immunization. Extract TS, extract ES and the cytoplasmic fraction of Raji cells at protein concentrations of 0.5, 0.6 and 3.2 mg/ml, respectively, were mixed with equal volumes of Freund's adjuvant. Initially, 1 ml of each of these mixtures were inoculated intramuscularly into different New Zealand white rabbits. These animals were subsequently boosted with 0.4 ml of the same anti-

865

gen mixtures at weekly intervals for two months. Hyperimmune and pre-immunization sera were obtained from individual animals by heart puncture and stored at --20 ° C until use. Serology. Agglutination and indirect membrane fluorescence tests were carried out concurrently. All reactions were carried out in minimal essential medium containing 1% fetal calf serum unless stated otherwise. To determine the serum agglutinin level, 0.2 ml of a serially diluted serum was mixed with an equal volume of cell suspension containing approx. 1 . 1 0 6 cells per ml and incubated at 37°C for 30 min. The mixture was then made up to 1 ml and the cell agglutination was read visually against a serum blank. Agglutination titers are defined as the reciprocal of the maximum serum dilution which gives visible agglutination. The same cells were then washed three times and reacted at 37°C for 1 h with a 1/32 diluted FITC-conjugated goat anti-rabbit immunoglobulin (Behringwerke AG, Germany), washed three times with phosphate-buffered saline and mounted in 50% glycerol. All immunofluorescence titers are expressed as the reciprocal of the maximum serum dilution giving positive immunofluorescence. Resul~

Selectivity of lactoperoxidase radioiodination Whole Raji cells were radioiodinated as described in Materials and Methods. Under the present conditions of labelling, over 90% of the total cell-bound acidprecipitable radioactivity occurred in the membrane fractions and the radiospecific activity of the latter greatly exceeded that of the corresponding cytoplasmic fractions (Table I). In view of the large difference in radiospecificities existing between these fractions, it is conceivable that enzymic radioiodination occurred exclusively at the cell surface and that the cytoplasmic radioactivity might be attributed to the radioactive products which were originally associated with the cellular membrane but were inadvertantly solubilized during the subsequent sub-cellular fractionation steps. These results are compatible with the observations of other researchers indicating that lactoperoxidase radioiodi-

TABLE I DISTRIBUTION OF ACID-PRECIPITABLE R A D I O A C T I V I T Y IN SUBCELLULAR F R A C T I O N S OF R A J I CELLS EnzymioAtlly labelled Raji cells were fzactionated i n t o m e m b r a n e a n d c y t o p l a s m as d e s ~ I b e d i n Materials and Methods. Total r a d i o a e t l v i t y recovered in these fractlo~s are e x p r e s s e d as p e r c e n t o f that o f w h o l e cells. Fraction

Whole cells Membrane Cytoplasm

Experiment 1

Experiment 2

Percent radioaethdty

Radiosl~ecifle activity ( c p m / m g protein)

Percent radioacUvtty

Raclioepecific aet/v/ty (ePmlmg protein)

100 98.4 5.2

6 328 11 780 840

100 91.0 2.2

22 286 47 014 I 046

OF RADIOACTIVITY

AND PROTEIN

IN EXTRACTS

AND SUB-CELLULAR

FRACTIONS

OF RAJI CELLS FOLLOWING

IONIC SHOCK

W h o l e cell TS ES Cytoplasm Membrane

Fraction

o f w h o l e cells.

100 8.0 22.0 7.9 41.4

100 2.5 6.5 27.9 32.0

8842 29118 37950 2519 11452

100 18.8 37.4 0.3 48.6

Radio activity (%)

Specific activity (cpm/mg protein)

Radioactivity (%)

Protein (%)

E x p e r i m e n t II

Experiment I

i00 9.9 7.1 26.7 33.0

Protein (%)

23463 48947 123664 2638 32917

Specific activity (cpm/mg protein) 100 4.9 8.7 1.2 64.8

Radioactivity (%)

I00 2.6 7.4 25.6 54.7

Protein (%)

E x p e r i m e n t III

22127 42028 25773 1007 25426

Specific activity (cpm/mg protein)

I00 10.6 22.7 3.1 51.6

Radio activity (%)

Average

I00 5.0 7.0 26.7 39.9

Protein (%)

C y t o p l a s m i c a n d m e m b r a n e f r a c t i o n s w e r e d e r i v e d f r o m R a j i cells a f t e r i o n i c s h o c k t r e a t m e n t . R a d i o a c t i v i t y a n d p r o t e i n c o n t e n t s axe e x p r e s s e d as p e r c e n t o f t h o s e

TREATMENT

T A B L E II DISTRIBUTION

O~

(30

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nation is specific for the Raji cell surface components. (Kennel and Lerner [5], Schmidt~Ullrich et al. [6]). Quantitative effects of ionic shock treatment Three batches of Raji cells similarly radioiodinated with lactoperoxidase were used to assess the extent of solubilization of cen surface components by ionic shock treatment. In these and the previous instances the extent of mdioiodination as determined by the radiospecific activities of the resulting labelled cells varied in individual experiments. Likewise, the extent of radioactivity released from different batches of labelled cells by ionic shock treatment also varied (Table II). This is believed to be due in part to a varied degree of exposure of those group which are susceptible to enzymic radioiodination, presumably reflecting the dynamics of association of the membrane components (Bretscher and Raff [ 7 ]). However, despite this experimental variation, it is apparent that ionic shock treatment resulted in extensive solubillzation of the radioactive products from the labelled cells. The radiospecific activities of the resulting extracts invariably exceeded that of the corresponding residual membrane (Table II). These results are considered as evidence of the efficiency of the ionic shock treatment in extracting the surface components from Raji cells. The alternative interpretation that enzymic labelling per se might have modified the susceptibility of the cells to ionic shock treatment seems unlikely as will be shown.

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F i g . 1. S t a i n e d polyacrylAmtde g e l e l e c t r o p h o r e t i e p a t t e r ~ o f t h e m e m b r a n e f r a c t i o n of R a j i e e l l l ( a ) before and (b) after ionic shock treatment.

II

868

Electrophoresis of extracts In order to study the qualitative effects of the ionic shock treatment the extracts and membrane fractions obtained from Raji cells before and after shock treatment were electrophoresed in sodium dodecylsulfate-acrylamidegel as described. The gels were stained with amido black before slicing for radioactivity counting in order that both the stained and the radioactivity patterns could be obtained from the same gel. The effects of ionic shock were not apparent from comparison of the stained patterns of the membrane fraction before and after these treatments. (Figs. l a and lb). However, the distribution of radioactivity obtained with the shocktreated membrane was distinct from that of the untreated membrane, suggesting that the radioactive products are differentially susceptible to ionic shock (Fig. 2). The stained sodium dodecylsulfate-gel patterns obtained with the extracts derived from four batches of enzymically labelled and one batch of unlabelled cells are depicted in Figs. 3 and 4. Whether or not the cells were radioiodinated, the Tris • HC1 extracts were shown to consist of five major polypeptides and

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Fig. 2. R a d i o a c t i v i t y patterns obtained from the same acrylamide gels as i n F i p . l a (top) and l b (bottom). Fig. 3. Stained polyaerylamLde gel electrophoretic patterns of TS e x t r a c t s derived from one b a t c h of unlabelled (ex treme right) and four batches of lactoperoxidase-labened Raft cells.

869

Fig. 4. Stained p olyacrylamide gel patterns of ES extrac t s from one ba t c h of unlabelled ( e x ~ e m e right) and four batches of lactoperoxidase-labelled Raji cells.

about fifteen minor ones. The major polypeptides were regularly detected in these and all the TS extracts studied thus far. The minor polypeptides were observed at varying frequencies, presumably depending on the concentrations of individual components occurring in different batches of TS extracts. ES extracts were likewise regularly shown to be made up of seven major polypeptides and a varying number of up to twelve minor components. It is apparent from these and other stained patterns that enzymic radioiodination does not appear to have altered the susceptibility of the cells to ionic shock treatment. The polypeptides identified in the stained patterns of the e x t ~ c t s derived from the four batches of enzymic labelled cells were compared by their electrophoretic migration. A total of 22 shock-released polypeptides were thus identified and were numbered in increasing order of electrophoretic migration. The frequencies of occurrence of the individual shock-released polypeptides in these TS extracts and ES extracts are shown in Table III). The majority of polypeptides of similar electrophoretic migration were d e ~ in both extracts. Polypeptides 7 and 11, however, appeared to occur exclusively in extracts ES and TS, respectively, while polypeptide 9a was only remlved t ~ m a single batch' of extract TS. Fig. 5 shows the electrophoretic migration of some of these polypeptides in relation to marker proteins of known molecular weight. The radioactivity patterns of the TS and ES extracts of one batch of the

870 TABLE III ELECTROPHORETIC ANALYSIS OF SHOCK-RELEASED POLYPEPTIDES F r e q u e n c y of d e t e c t i o n was d e t e r m i n e d b y electrophoreti e an~dysis of four different b a t c h e s o f extracts from lactoperoxldase-radioiodinated Raji cells (see t e x t for details). Numbet

1 2 3 4 5 6 7 8 9 9a 10 11 12 13 14 15 16 17 18 19 20 21

Migration +- S.E. (ram)

2.93 4.60 7.40 9.30 10.29 12.50 13.63 15.31 18.88 21.00 24.50 28.33 30.31 35.13 39.56 42.79 47.21 49.83 54.57 59.50 62.33 66.50

± 0.17 -+ 0.19 -+ 0.19 -+ 0.16 -+ 0.28 -+ 0.16 ± 0.43 ± 0.16 +- 0.16 ± 0.31 -+ 0.44 + 0.34 ± 0.35 ± 0.38 ± 0.47 ± 0.42 ± 0.44 ± 0.35 ± 0.76 ± 0.33 ± 0.76

Frequency of d e t e c t i o n by staining TS

ES

(TS + ES)

214 214 1/4 2/4 3/4 2/4 0/4 4/4 4/4 1/4 4/4 3/4 4/4 4/4 4/4 4/4 4/4 2/4 4/4 1/4 3/4 2/4

4/4 3/4 4/4 3/4 4/4 2/4 4/4 4/4 4/4 0/4 4/4 0/4 4/4 4/4 4/4 3/4 3/4 1/4 3/4 1/4 1/4 1/4

6/ 8 5/8 5/8 5/8 7/8 4/8 4/8 8/ 8 8/8 1/8 8/8 318 8/8 8/8 8/8 7/8 7/8 3/8 7/8 2/8 4/8 3/8

* * * * *

* * * * * * *

F re que nc y of detect i on b y radioactivity (TS + ES) 318 418 7/8 2/8 7/8 3/8 4/ 8 8/8 0/ 8 8/8 8/8 0/ 8 7/8 2/8 4/ 6 7/ 8 7/8 5/8 5/8 3/8 5/8

* Major p o l y p e p t i d e s

radiolabelled cells are shown in Figs. 6a a n d 6b, respectively. It was evident f r o m these and the radioactivity patterns obtained with the other batches of extracts that both the TS and ES extracts were extensively radiolabelled, and consisted of radioactive products with a wide range of electrophoretic mobilities. However, the complexity of these radioactivity patterns only allowed a tentative assignment of the radioactive peaks to individual or groups of polypeptides on the basis of their respective electrophoretic mobilities. The frequencies with which the shock-released polypeptides might thus be accounted for in t h e radioactivity patterns of these extracts are shown in Table III. Only two polypeptides, i.e 9 and 12, appear to be inaccessible to enzymic radioiodination and are consistently unaccounted for in the radioactivity patterns. The remaining polypeptides appeared to be radioiodinated and might be ascribed to various radioactivity peaks at the indicated frequencies.

Immunological studies of extracts Sera from rabbits immunized with extracts TS and ES reacted with live Raji cells giving intense indirect membrane immunofluorescence (Fig. 7). The same sera also a~glutinated live Raji cell efficiently such that their agglutinated titers are comparable to the corresponding membrane immunofluorescence titers. By contrast, the anti-cytoplasmic rabbit serum had no detectable agglutinating

871

Fig. 5. Distribution of shock-released polypeptides in extracts TS and ES. Corresponding p o i t i o n s of marker p r o t e i n s are shown: BSA, bovine serum albumin; H and L chain, heavy and light chains of h u m a n 7-globulin, respectively ; OVA, ovalbumin; TRY, trypsin.

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Fig. 6. Rad/oaetivlty' patterns of (a) TS0 and (b) ES derived from lactoperoxidase-labened Rall cells.

872

Fig. 7. Indirect i m m u n o f l u o r e a c e n c e staining of RaJi c e l k using h y p e r i m m u n e rabbit sera to e x t r a c t T S or extract ES and F I T C - c o n j n g a t e d goat anti-rabbit i m m t m o g l o b u K u s .

T A B L E IV MEMBRANE

REACTIVITY

OF RABBIT

SERA

IMMUNIZED

AGAINST

RAJI

CELL

EXTRACTS

G e o m e t r i c m e a n titers w e r e c o m p u t e d f r o m t h e resulta of at least three separate titratiorm b y t h e indlzect m e m b r a n e i m m u n o f l u o r e s c e n e e or agglutination m e t h o d tu~ag llve Raji cells as described in Materials and MethOds. Sera

A. a n t i - e x t r a c t TS B. a n t i - e x t r a c t TS C. a n t i - e x t r a c t ES D. a n t i - e x t r a c t ES E. a n t i - c y t o p l a s m Non-immune

G e o m e t r i c m e a n titers Membrane i m m u n o f l u o r a s c e n c e

Agglutination

1380 3800 596 2150 35 <: 5

670 496 366 1200 ~5 ~5

activity and gave weak membrane immunofluorescence at low serum dilution (Table IV). Discussion In experimental animals, tumour-specific transplantation antigens are recognised by their ability to confer specific protection on the host against subsequent challenge with live tumour cells. These antigens are believed to be surface localized (Harris et al. [8]. Davis et al. [9]). However, in similar studies with human tumours, it is not conceivable that the same criteria can be applied operationally. Consequently, we have adopted the operational definitions that crude extracts containing tumor-specific transplantation antigen-like antigens of human origin (a) will elicit greater in vitro cell-mediated immune responses

873 in patients with the corresponding tumor, and (b) are localizedat the sur: face of the tumour cells. W e have obtained extracts from Raji cells which satisfied the fn~t criterion [1]. The present work was initiatedto determine the c e l l u l ~ localization of the components o f these extracts. In order to facilitate direct evaluation of the effects of ionic shock t r e a t m e n t on the surface components, we employed enzymically radioiodinated RajFcells. the validity of this experimental approach is evident from the present observations and those of the other workers (Kennel and Lerner [5], Schmidt-Ullrich et al. [6] ). that lactoperoxidase radioiodination occurred at the cell surface and that enzymic labelling per se did not appear to have changed the susceptibility of the cells to subsequent ionic shock treatment. The results indicate that ionic shock treatment is an efficient method for extracting components from the surface of Raji cells resulting in the solubilization of an average of approx. 30% of the total cell-bound radioactivity. As t h e cytoplasmic components were not radioiodinated to any significant extent their occurrence in the extracts would be expected to lower the radiospecificity of the latter relative to that o f t h e membrane or of the whole labelled cells. The findings t o the contrary that the radiospecific activities of the extracts consistently exceeded that of the corresponding membrane suggest t h a t the former COnSist largely of cell surface com~ ponents. This is supported by the results of the subsequent electrophoretic studies from which, while the stained and radioactivity gel patterns could not be unequivocally correlated, it is apparent that the majority of the shockreleased polypeptides, as identified in the stained patterns, appear to be accessible to enzymic radioiodination. The results of the studies with hyperimmune rabbit sera indicated that the anti-extract TS and anti-extract ES sero-activity are directed against the cell surface (Fig. 7), and therefore corroborate the results obtained by lactoperoxidase radioiodination of Raji cells. In contrast, sera from rabbits previously immunized against the cytoplasmic fraction only displayed reactivity to the intracellular components of Raji cells and were not reactive against the cell membrane even at low serum dilution. Subsequent studies with these sera indicated further that the reactivity of the anti-extract ES sera was absorbed more extensively by different human red blood cells than was that of the anti-extract TS sera, suggesting that extracts TS and ES are antigenically distinct (unpublished results). These findings supports the results of the electrophoretic analysis of the extracts. The present studies formed the basis for further purification of factor(s) responsible for eliciting in vitro cell-mediated immune responses in nasopharyngeal carcinoma patients. A total of 22 shock-released polypeptides were identified in TS and ES, but only two, i.e. 9 and 12, appeared to be consistently inacceasible to enzymic labelling. The contention that these two polypeptides may be non-iodinated because they lack tyrosine rather than not being on the cell surface is not likely because when uniformly radioiodinated extracts were electrophoresed radioactivity peaks with migration characteristics similar to those of polypeptides 9 and 12 were resolved (unpublished results). As the in vitro cell-mediated immunity
874

Acknowledgements This work was supported by the Hong Kong Anti-Cancer Society, University of Hong Kong Committee for I~AgherEducation and Research and by a collaborative research agreement between the Biological Carcinogen Unit, IARC, Lyon and The Department of Microbiology, University of Hong Kong under Contract No. 1 CP 43296 within the Virus Cancer Program of the National Cancer Institare, U.S.A. References 1 Ng, W.S., Ng, Mun, H., Lo, E.H.M., Ho, H.C., Chou, J. and LameUin, J.P. (1976) s u b m i t t e d to J. Natl. Cancer Inst.

2 3 4 5 6

Cone, R.E. and Marchalonis, J.J. (1973) Ajebak 51 (Part 5), 6 9 9 - - 7 0 0 Naehman, R.L., Hubbard, A. and Ferris, B. (1973) J. BioL Chem. 248, 2928--2936 LowTy, O.BL, R o u b r o u g h , N.J., F a ~ , A.L. and R a n d a l l R.J. (1951) J. Biol. Chem. 193, 265--275 Kennel, S.J. and Lemer, R.A. (1973) J. MoL Biol. 76, 4 8 5 - - 5 0 2 SchmidtoUlrich, R., Ferber0 E., Knilfermann, H., Fischer, H. a n d Hoelzl Wallach, D.F. (1974) Biochim. Biophys. Aeta 332, 175--191 7 Bretscher0 M.S. a n d Raff, M.C. (1975) Nature 258. 43--48 8 HaxrJs, J.R., Price, M.R. and Balwin, R.W. (1973) Bioehlm. Biophys. Acta 311, 600--614 9 Davis, I.AB.I., Nicldin, M.G, and Augustln, R. (1975) Nature 256, 49--50