the vol. no. 12, issue of june 25, pp. 7751-7766,1983 … · monoclonal antibodies to...

THE J O U R N A L OF BIOLOGICAL CHEMISTRY

Printed in 1l.S.A. Vol. 258, No. 12, Issue of June 25, pp. 7751-7766,1983

Monoclonal Antibodies to Tissue-specific Chromatin Proteins* (Received for publication, March 14, 1983)

Jeff N. VanderbiltS and John N. Anderson From the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Antisera raised in mice to chromatins from different tissues of the chicken reacted preferentially with the chromatin type that was used for immunization. This tissue specificity was also evident in the spectrum of monoclonal antibodies generated when mice were immunized with erythrocyte chromatin. Three erythroid- specific antigens and one antigen that was present in a number of chicken tissues were characterized in fur- ther detail. These antigens, which comprised less than 0.1% of the erythrocyte chromatin proteins, were nuclear localized although three were also detected in the cytoplasm. Two of the erythroid-specific antigens existed as multiple polypeptides in isolated chromatin. The multiple chromatin forms of one antigen were derived from a precursor protein that was selectively cleaved within 1 min after erythrocyte lysis. Analysis of this antigen in extracts from erythrocytes and reticulocytes indicated that the cleavage of the precursor protein was developmentally regulated in vivo.

The nonhistone class of chromosomal proteins is a complex mixture containing more than 500 distinct polypeptides (1, 2). Largely because of this heterogeneity, progress toward understanding the function of individual nonhistone proteins has been slow and will ultimately require highly specific probes for their quantitation and isolation. In this regard, several laboratories have recently reported the generation of monoclonal antibodies that recognize nonhistone chromatin proteins (3-10). A major advantage of this approach (11) is that antibodies to specific proteins can be obtained following immunization with a complex antigen mixture such as chromatin.

Historically, chromatin and its components have served as antigens for the production of a wide variety of antisera (12, 13). Antisera directed against the nucleosomal core histones react with the corresponding histones from many different tissues and species, presumably reflecting the conserved nature of these basic proteins (14, 15). Antisera to nonhistone proteins or dehistonized chromatin, in contrast, display both species and tissue specificity (13, 16-27). This specificity is particularly striking since electrophoretic studies have revealed few differences among the detectable nonhistone chromatin proteins in different tissue types (28-30). Thus, the immune system apparently selects for the tissue-specific proteins of chromatin. Because this protein subset may include members that play a role in regulating genes that are ex-

* This work was supported by Grant R01-CA25799-03 from the National Cancer Institute and a grant from the Purdue Cancer Center. The costs of publication of this article were defrayed in part by the Payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

# Supported by a National Institutes of Health Predoctoral Train- eeship.

pressed in a tissue-dependent manner, monoclonal antibodies to these proteins could provide powerful tools for the study of gene function in eukaryotes. In previous studies, we described the preparation of a library of monoclonal antibodies to proteins associated with hen oviduct chromatin (9,101. Eighty per cent of these antibodies recognized proteins that were found in the hen oviduct but not in hen liver, lung, or erythrocytes. In this report, a similar approach was taken to prepare antibodies to proteins associated with chicken erythrocyte chromatin. Four of these antigens were then characterized with respect to their tissue distribution, apparent molecular weight and subcellular location.

EXPERIMENTAL PROCEDURES'

saline I rovgh the l . f tven??icTpriorlo removal of nonerythroid tissues. P z e r t i o n of Nuclei and Chromatin. * i r e LeShorn hens were pereuocd with

Nuclei from these tiesues were prepared essentially DB descrlbed previously (31.32). Tro dif ferent methmda were employed for preperation of erythroryre and reticulocyte nuclei from no-1 and anemlc ( 3 3 ) hems. In arthod 1. erythroid nuclci rere prepared 08 described by Yelnfraub and Croudlne 04). *ebod I1 was

Xane st. ( 7 ) . Mtclei pcepeed by f h l s athod e r e devold of v l r l b l a performed accnrdlng I o the procedure of Buach end Smcrana (15) as modified hy

cytopl~~mlc Contamlnalion when enlmlned by electron microscopy. For the isolation yf chr-tln. ouclel e r e lysed by haogeniEstlon in NET (80 rn Hac1.

centrifuged at lO.Om "8 for 5 .In and rhe pellets were suspended in SET (150 d 4 10 M WTA . 5 d( n l s , 5 n+~ sadxm b c s u l f i t e . ?H 6.5). me homogenarr

zt moa cemplrelure. Pollorlng another wter rash, 100 Yl af anrlbwdy so lut ion were IOcvbafed In the wells of the microlitel plates for 2 h at rocm temperature.

antisera were dilvced I l l 0 0 1" PBS, 1 1 BSA. M:er I later wash to remow= unbound Hybridnns culture supernatsots were diluted 114 and ascite@ f l u i d 01 D Y S ~

of ~ I-rabblt a n t h a u e e IgC (100 111) In each wel l . Finally, the plates were sntf93ay. rhe plates were Incubated for 2 h 'IC room temperature r i c h 20,000 CI.

washed with PIS and the lodivldual wells were separated from each other and the bound radioact CY determined. Ilebblf anti-mouse k c anriaere (Cappell m e labelled with "'1 by P chloramine-T procedure rhlle b u n d to a Sepbrosc-use l@ imvnosorbent (171. Following elutloo from tho Sepharose-use I&. th is teasent had m approximare apeclfic accLri ty of 500 c p l n g .

. . ,

Portions of this paper (including "Experimental Procedures" and Table I) are presented in miniprint as prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 83M-0683, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

* The abbreviations used are: EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(8-aminoethyl ether)-N,N,N',N'-tetraa- cetic acid; PMSF, phenylmethylsulfonyl fluoride; NP40, Nonidet P-40.

7751

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7752 Tissue-specific Chromatin Proteins

i n PBS-11 8%. washed again with PBS. mounted ln glycerol (pH 9). and eramined fluorescein lso,hiocvanate-conjussied rabbit aoti-mouse I g C ( s l p a ) diluted 1/10

by phase contrast and flvoreacence miicroscopy. Uekgound staining was assessed for each fixation procedure wlfh sscifes fluid from the IgC-SecrrlinB Weloma. This backgound s l a l n i n g is show in the bottom center Panel of Flg. 6 On a fixed by Herhod 8. Back~round sfalnlnl by the other nethods gave almilar r.lUltS.

Cellular Fractionation. All ce"lr1f"gatlon steps e r e fur 5 d" a r 10,000 ' 6 . ~ry-8-n RSB, 0.1% W-60 (RSB: IO .M NaC1, I d WgCl LO *I Trl~. pH 7 . A ) , centrifuged. and the supernatant was removed as the CYC&IaSmlC f r a c t l m . me nuclear pellet "as washed vlfh RSB. NET and SET and the f lne l

of chromatin wag b o i l e d in SDS electrophoretic ~ampple buffer. nie ~ u p e r n a r l n r s from the NET and SET extractl~ns are designated NET-nucleoplasmic and SET-nucleoplaralc fractions. Table 1 shows the concentrsfions of the antigens in these f r a c l l u n r re lat ive Lo fhetr concentratlunr In rhe unfraeclonaled erythrocyte. In this experiment, 20 Y8 of prote in from each fraction r e r e electrophoresed. imunoblolted and the re lat ive ~ m ~ e n l r a t l o m of the ant igens determined by degnltonerric scanning of Lhe auforsdlogrsma. In Plg. b,6pcoteins fram about 3 X IO cello or from the cell Fra~CLonr derlued from 3 X

the gel lanes sere 110. 09, 9, 6 and 22 up, far unfractlonated erphrocyres . 10 ceiir. _re electrophoresed on 152 gels . The amount6 of p m ~ e l n appl ied t o

cytoplas~lc. NET-nucleoplasmic, SET-nucleoplasmic and chromatin frarrlons. respeCll"elY.

RESULTS AND DISCUSSION

Rabbit anti-chromatin antisera displaying tissue specificity for the immunizing chromatin have been described by several laboratories (13, 16-27). Similar findings are shown in Fig. 1 for four different mouse antisera, each generated against chromatin prepared from one of four different chicken tissues. The serum from each immune mouse was tested against the four chromatin preparations by solid phase radioimmune assays. For each antiserum, the highest reactivity occurred on the chromatin type that was used for immunization. Results similar to those shown in the figure were obtained when chromatin proteins, free of DNA, were assayed with the four antisera." These data appear to reflect an antibody response directed principally toward tissue-specific chromatin proteins even though few tissue-specific stained protein bands in these chromatin samples were detected by one-dimension SDS-gel electrophoresis.3

Our previous results demonstrated that most of the monoclonal antibodies obtained from mice immunized with hen

J. N. Vanderbilt and J. N. Anderson, unpublished observation.

a

6

4

2

0

8

6

4.

2.

X I OVIDUCT IMMUNE 4 LIVER IMMUNE / d / 1

LUNG IMMUNE IiMMUNE P ERYTHROCYTE

I

011 I :o ai, I 011 1.0

CHROMATIN (A26o/ml)

FIG. 1. Production of tissue-specific chromatin antisera in the mouse. The indicated amounts of soluble chromatin from hen oviduct (x), liver (01, lung (A), or erythrocyte (0) were incubated in the wells of microt.iter plates. The plates were incubated with a Koo dilution of antisera from mice immunized with chromatin from the indicated tissues, then with Iz5I-rabbit anti-mouse IgG, and the bound radioactivity was determined. Nonspecific binding was determined by incubation with a %cm dilution of preimmune sera in place of the immune sera. The levels of nonspecific binding, which represented no more than 10% of the binding of the immune sera on the immunizing chromatins, were subtracted from all data points in the figure.

oviduct chromatin recognized oviduct-specific chromatin proteins (9, 10). Since tissue-specific anti-chromatin antisera were produced in mice irrespective of the tissue source used for immunization (Fig. l), monoclonal antibodies recognizing tissue-specific proteins associated with chromatin from tissues other than the oviduct should also be easy to obtain. Spleen cells from mice immunized with hen erythrocyte chromatin were fused with myeloma cells and the hybrids were screened for anti-chromatin antibody production by solid phase radioimmune assays. A total of 207 hybrid cell lines that produced antibodies reactive with erythrocyte chromatin were recovered from two separate fusions. A large fraction (86%) of these antibodies reacted preferentially with erythrocyte chromatin as compared to chromatins from oviduct and liver." Selected cell lines were subcloned on agar and amplified as ascites tumors, and their antibodies were ana- lyzed in more detail as described below. These antibodies were chosen a t random from a group of 77 whose antigens could be detected by immunoblotting analysis.

Fig. 2 shows the tissue distribution of the antigens recognized by four antibodies as revealed by immunoblotting analysis. The protein (RBC 13) recognized by one antibody (anti- RBC 13) was present in all tissues examined. In contrast,


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Tissue-specific Chromatin Proteins 7753

STAINED RBC 1 RBC 2 R E O K L C R E O K L C R I O K L C

FIG 2. Detection of four chromatin antigens. Chromatin proteins from hen reticulocytes ( R ) , erythrocytes ( E ) , oviducts ( O ) , kidneys ( K ) , livers ( L ) , and crops ( C ) were separated on six 15% SDS-polyacrylamide gels. The proteins in one gel were stained with Coomassie blue (upper left panel) while those in the others were transferred to nitrocellulose sheets. Four sheets were probed with monoclonal antibodies in ascites fluid for detection of antigens (RBC 1, RBC 2, RBC 11, and RBC 13), while one sheet (control) was probed with ascites fluid from an IgC-secreting myeloma. The molecular weights of the reacting polypeptides were 93,000-230,000; 87,000; 87,000-210,000; and 78,000 for RBC 1, RBC 2, RBC 11, and RBC 13, respectively.

. n

RBC 11 RBC 13 CONTROL K L C R E O K L C R E O K L C R E O

three antibodies detected protein antigens (RBC 1, RBC 2, and RBC 11) that were present in the reticulocyte and erythrocyte chromatin preparations but absent in chromatin from the other chicken tissues. Similarly, these proteins were not detected in chromatin from nonerythroid cells by solid phase radioimmune assays nor were they detected when the immunoblots shown in Fig. 2 were exposed for longer periods of time." These three antigens were also present in mature erythrocytes of the primitive erythroid lineage from 6-day-old chick embryos.3 A study of younger embryos is required to determine when they first appear during erythroid develop- ment. It is possible that RBC 1,2, and 11 are actually present in nonerythroid cells but are not associated with the chromatin. This possibility is unlikely, however, since none of these proteins were detected on immunoblots of total tissue homogenates from nonerythroid tissues but all were detected in homogenates from erythrocytes.:' In addition, anti-RBC 1, 2, and 11 did not stain oviduct and liver cells when frozen sections from these tissues were examined by indirect immunofluorescence microscopy, whereas nuclei within these cells fluoresced brightly when anti-RBC 13 was employed in the analysis:'

Antibodies RBC 1 and 11 detected multiple discrete bands on immunoblots of chromatin proteins (Fig. 2). To study the mechanism responsible for this phenomenon, freshly drawn blood or isolated erythrocyte chromatin was placed directly into boiling SDS-electrophoretic sample buffer and applied to 7.5% SDS gels, and blots prepared from these gels were probed with anti-RBC 1 and anti-RBC 11 (Fig. 3). Anti-RBC 11 defined the same series of protein bands in whole blood and in erythrocyte chromatin. Other investigators have attributed similar observations to the recognition of several functionally or evolutionally related polypeptides by a given monoclonal antibody (6, 7). Anti-RBC 1 detected a major ( M , = 230,000) and a minor ( M , = 213,000) band in the intact erythrocyte from the whole blood extract. In the chromatin sample, however, these bands were replaced by two smaller molecular

RBC I I RBC I A B A B

e213 k + 1 9 4 k

93 k

FIG. 3. Detection of RBC 1 and RBC 11 in extracts from intact erythrocytes and erythrocyte chromatin. Hen blood ( A ) and erythrocyte chromatin ( B ) were boiled in SDS-electrophoretic sample buffer and electrophoresed on 7.5% polyacrylamide gels. Im- munoblots were probed with anti-RBC 1 or anti-RBC 11.

weight fragments ( M , = 194,000 and 93,000) suggesting that RBC 1 was selectively cleaved during chromatin isolation. Fig. 4 shows the time course of the formation of these fragments in erythrocyte (top panel) and reticulocyte (bottom panel) lysates at 4 "C. Within !4 min after erythrocyte lysis, the two additional lower molecular weight fragments appeared in the lysate. These bands increased in intensity with time


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until, a t 5 min after lysis, a stable pattern similar to that of isolated chromatin was apparent. The sequential breakdown of RBC 1 also occurred in the reticulocyte lysate, but its rate was about lO''-fold slower than in the lysate from the erythrocyte. The breakdown of RBC 1 in the erythrocyte lysate was a highly selective process since it was not accompanied by a change in the molecular weight of the polypeptides recognized by anti-RBC 2, 3, and 11 nor by a change in the Coomassie blue staining profiles of the erythrocyte homoge- nate polypeptides as revealed by electrophoretic analysis:' The in vitro proteolysis of RBC 1 in the erythrocyte lysate was unaffected by p-aminobenzamidine (1 mM), PMSF (1 mM), aprotinin (1 unit/ml), bacitracin (0.1 mM), or pepstatin A (10 p ~ ) but was completely inhibited by inclusion of leu- peptin (0.15 mM) or a mixture of EDTA (10 mM) and ECTA (2 mM) in the incubation buffer.:'

The rapid and selective proteolysis of RBC 1 during in vitro incubation of the erythrocyte lysate raises the possibility that the cleavage of this protein occurs in the intact cell. The presence of the two forms of RBC 1 in SDS extracts from intact erythrocytes (Fig. 4, zero time sample) and whole blood (Fig. 3) supports this view. Presumably, the larger form of RBC 1 ( M , = 230,000) is the precursor to the smaller form ( M , = 213,000) in vivo since the smaller species increases a t the expense of the larger one during the 1st min after cell lysis (Fig. 4, top panel). It is also interesting to note that the smaller molecular weight form of RBC 1 is prominent in the extracts from the intact erythrocyte (Figs. 3 and 4) but is barely detected in the immunoblots from reticulocytes (Fig. 4). This relationship is more clearly illustrated in Fig. 5 which shows the relative proportion of the two presumptive in vivo

TIME (mid

0 1/4 1 / 2 I 2 5 20 60 le0 900

Fw. 4. Sequential breakdown of RBC 1 in erythrocyte and reticulocyte lysates. Erythrocytes (top panel) and reticulocytes (bottom panel) were lysed by suspension in 0.5% NP40-RSB buffer and incubated for the indicated times a t 4 "C. Samples were electrophoresed on 7.5% gels, blotted, and probed with anti-RBC 1. Only the portions of the autoradiograms that contained bands are presented in the figure.

forms of RBC 1 in erythroid cells during the course of phenylhydrazine-induced anemia in a single chicken. In the mature erythrocyte from the pretreated hen, approximately 30% of the protein recognized by anti-RBC 1 existed in the lower molecular weight form (lane A ) in agreement with the results presented in Figs. 3 and 4. Following five daily injections of phenylhydrazine, this value decreased to about 5% (lane C) and, at this time, more than 90% of the cells in the circulation were reticulocytes. Maturation of the reticulocytes into erythrocytes upon cessation of phenylhydrazine treatment was accompanied by an increase in the smaller molecular weight species of RBC 1 (lanes D-G). These results, and those shown in Figs. 3 and 4, suggest that the lower molecular weight species of RBC 1 in the erythrocyte is derived from the larger one by a developmentally regulated in vivo processing event. An understanding of the biological significance of this processing step awaits a description of the function of RBC 1 in erythroid cells.

Two different approaches were taken to examine the subcellular distribution of the erythrocyte antigens. In the first (Table I and Fig. 6, left panel), intact erythrocytes and cytoplasmic, nucleoplasmic, and chromatin fractions prepared from these cells were solubilized and electrophoresed, and the antigens were detected by the immunoblotting procedure. All antigens were preferentially associated with the chromatin fraction since their concentrations in this fraction were 3.5- 7-fold greater than their concentrations in the intact erythrocytes (Table I ) . Protein antigens RBC 11 and RBC 13 were exclusively chromatin localized whereas RBC 1 and RBC 2 were detected in both cytoplasmic and chromatin fractions (Table I and Fig. 6). The only RBC 1 polypeptide detected in the cytoplasmic fraction was the M, = 93,000 fragment that was presumably generated by the proteolysis of the parent M , = 230,000 species (see Fig. 4). This fragment may have been released from the nuclei following its cleavage from a larger chromatin bound protein.

Also shown in Fig. 6 are indirect immunofluorescence mi- crographs of blood smears stained with each of the monoclonal

A B C D E F G

FIG. 5. RBC 1 in erythroid cells during phenylhydrazine- induced anemia. A hen received five daily injections of phenylhydrazine. Blood samples, collected directly into SDS-electrophoretic sample buffer a t 0, 2, 5, 7, 9, 12, and 22 days after the first injection (lanes A-G, respectively), were electrophoresed on a 7.5% gel, blotted, and probed with anti-RBC 1. The percentage of reticulocytes in blood samples A-G, as revealed by Giemsa staining, were <1, 26,94,6,<1, < I , and <I, respectively. Only the portion of the autoradiogram that contained bands is presented in the figure.


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Tissue-specific Chromatin Proteins 7755

FIG. 6. Subcellular location of the antigens. L e f t panel, unfractionated erythrocytes ( A ) or cytoplasmic ( B ) , NET-nucleoplasmic ( C ) , SET-nucleoplasmic ( I ) ) , and chromatin ( E ) fractions were isolated as detailed under “Experi- mental Procedures” and electrophoresed on 15% gels. Immunoblots were probed with the antibodies for detection of the indicated antigens. The control was the same as in Fig. 2. Only the portions of the autoradiograms that contained bands are presented in the figure. Middle and right panels, blood smears were stained with the indicated antibodies and viewed by phase-contrast (right panel) and fluorescence (middle panel) microscopy.

RBC I

RBC 2

RBC It

RBC 13

CONTROL

A B C

e - I)-

L -.

D E

t

4 c. L

I .

antibodies. Nuclear staining was found with all antibodies in agreement with the results of the cellular fractionation studies discussed above. Weak diffuse cytoplasmic fluorescence was also detected with antibodies 1, 2, and 11 whereas RBC 11 was not detected in the cytoplasmic fraction by immunoblotting analysis. This discrepancy may have resulted from nuclear leakage of RBC ll during fixation of the erythrocytes or from failure to detect low levels of RBC 11 in the cytoplasmic fraction by the immunoblotting procedure. Although each antibody required a different procedure for optimum fluorescence (see “Experimental Procedures”), none of the procedures tested produced subcellular patterns that were radically different from those shown in the figure.3 Immuno- fluorescent staining of reticulocytes from anemic hens also revealed no differences in the subcellular occurrence of any of the antigens when compared to the mature erythrocyte.3

During the preparation of this manuscript, Kane et al. (7) also described a series of monoclonal antibodies that reacted preferentially with chick red blood cell nuclear proteins. Their results, as well as those presented here and elsewhere (9, lo), clearly show that a large fraction of the monoclonal antibodies generated after immunization with unfractionated chromatin proteins or chromatin is directed against tissue-specific deter- minants of the nucleus. The erythrocyte antigens characterized in this report are minor components of the chromatin with each comprising between 0.01-0.12% of the total nonhistone proteins (see “Experimental Procedures”). These proteins, however, are apparently highly immunogenic in the mouse since multiple clones producing antibodies to each of them were recovered from two separate fusions.:’ Many of the predominant nonhistone proteins probably play a common structural or enzymatic role in different organisms and thus, like histones, may be poor immunogens because they are highly conserved in eukaryotes. Tissue-specific chromatin proteins, in contrast, may also be species-specific and therefore such proteins from the chicken may be highly antigenic in the mouse. These speculations aside, the results of this paper and the above mentioned studies demonstrate that

monoclonal antibodies against tissue-specific chromatin proteins are readily attainable by standard procedures.

Acknowledgments-We thank Bonnie Germain for her skillful technical assistance, the late Dr. David Evers for electron microscopy analysis, and Dr. Joann Otto for the use of her fluorescent microscope and instructions on its operation.

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J N Vanderbilt and J N AndersonMonoclonal antibodies to tissue-specific chromatin proteins.

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