Jugates. These Immunogens Comprise 2,4-Quinazolinedione-6-Azo-, Pseudouridine-, and Dihydrouridine-Albumins

ANTIBODIES TO HAPTEN-CONJUGA TED PROTEINS WHICH CROSS-REACT WITH RNA BY MERYL H. KAROL* AND STUART W TANENBAUM

DEPARTMENT OF MICROBIOLOGY, COLUMBIA UNIVERSITY COLLEGE OF PHYSICIANS AND SURGEONS Communicated by E. L. Tatum, December 19, 1966 In continuation of studies on antibodies which react with informational macro- molecules,1 the experimental induction with hapten-conjugated proteins2-5 of antisera capable of RNA cross-reaction has been attempted with several novel conjugates. These immunogens comprise 2,4-quinazolinedione-6-azo-, pseudouridine-, and dihydrouridine-albumins. Ancillary antigens containing uridine substituents6 were also prepared. ey-Globulin fractions derived from rabbit antisera to these conjugates react with RNA by precipitation, in confirmation of the results of Sela et al.6 with antigenic uridine-polypeptides. A uridine-specific serum, and the globulin fraction from a dihydrouridine antiserum, can also be shown to react with several RNA's by complement fixation tests. Antibodies to the uridine, pseudouridine, dihydrouridine, and quinazoline 2,4-dione determinants exhibit precipitation in gel with appropriate homoribopolymers and cross-react with denatured calf thymus DNA. This report describes antigen syntheses and the properties of their antisera with regard to RNA reactivity. Materials and Methods.-Uridine (U)- and Pseudouridine (4,)-conjugates: Uridine was oxidized to uridine-5'-carboxylic acid,',7 and was coupled to bovine serum albumin (BSA) and to rabbit serum albumin (RSA) by Boissonnas' mixed anhydride methods in dimethylformamide-water. Purification and isolation of the conjugated proteins was carried out as described earlier.2, 3 Spectrophotometric analyses2. 3 indicated that ca. 14 nucleosides were conjugated per mole of albumin (U-BSA, U-RSA; mol wt 70,000). 4, was oxygenated,7 the catalyst was removed, and the filtrate volume was reduced. After treatment with Amberlite IR-120 (H+), decantation, and evaporation of water, the liberated yellow oil crystallized rapidly (yield, 80%). o-5'-Carboxylic acid was recrystallized from methanol to give an analytically pure sample, mp 234.50 d., 0.01 M Tris-HC1 pH 7.4 = 263 mar, e = 7440). Coupling with albumins was carried out as described above. The conjugates contained ca. 10-11 haptenic groups per mole. "Reduced-uridine" (red. U)-conjugates: To 30 mg of U-BSA in 10 ml 0.1 M Na2HP04 7H20, 1 N NaOH was added to pH 9.2. After addition of 17.5 mg NaBH4, the flask was irradiated (G.E., germicidal lamp) and was stirred for 6.5 hr.9 Dialysis followed by lyophilization gave a white powder (Xm O.1M phosphate pH 6.8 = 267-270 mj). Spectrophotometric comparison with the parental conjugate indicated 70% reduction (ca. 10) of uridine residues. This estimate was confirmed by a direct assay for ureido groups after alkaline cleavage.10 Reduction of 11 of the 16 uridine groups on U-RSA occurred similarly. BSA and RSA were each irradiated in the presence of NaBH4 to yield altered carrier proteins (red. BSA and red. RSA). Quinazoline-2,4-dione-6-azo-proteins and -haptens: Nitration of benzoyleneurea gave the 6- nitro derivative,11 which was hydrogenated with Adams' catalyst in glacial acetic acid to 6-amino- quinazoline-2,4-dione. Addition of HCl precipitated the salt which crystallized from ethanol- water (4/6), mp above 3300 (49% yield). Diazotization and conjugation to BSA and to human serum albumin (HSA) followed a published method12 (Fig. 1). Spectrophotometric examination of these conjugates (Xmah 0.2 M phosphate pH 7-4 = 345 myA) indicated substitution mainly as monoazotyrosine derivatives (e, ca. 22,000).13 From the OD's, approximately nine residues of hapten were calculated per mole of the conjugates (Q-BSA and Q-HSA). The quinazoline-2,4-dione-monoazo- phenols were purified by repeated base-acid precipitation and, when possible, by further recrystal- lization from hot water. Jmmunochemical techniques: Rabbits were immunized and sera were collected according to 713 Downloaded by guest on September 24, 2021 714 BIOCHEMISTRY: KAROL AND TANENBAUM POPROC. N. A. S.

o 0 ~ N HNO3H,/it,(~,Na [ProteinHoNo]__

O HO II ~~~~~~0 N NN=N ii ~ CHi-CH-\f Protein H FIG. 1.-Synthesis of quinazoline 2,4(111,311)-dione- azo-protein conjugates.

established procedures."4 Bleedings from individual rabbits were pooled, and the globulin fractions were prepared.'8 Quantitative precipitin and hapten inhibition techniques were carried out as described by Kabat;16 specific precipitates were analyzed by a microbiuret technique.'7 Complement-fixation tests'8 were carried out in Tris-HCl saline (0.01 M Tris, 0.14 M NaCl; TBS) pH 7.45-7.55.14 Double diffusion procedures in gel'9 were performed on 1' X 3" glass slides coated with 2.5 ml 1% Noble agar (Difco) in 0.5 M glycine-0.14 M NaCl solution. Antigens and haptens were added in TBS. Gel hapten inhibition tests were carried out as previously described.20 Ribonuclease activity in immunoglobulins was determined by Spahr's method2' with C'4-poly U. Polyribonucleotides, DNA, and RNA: Calf thymus DNA (Nutritional Biochemicals, 1 mg per ml in 0.15 M NaCl-0.015 M Na citrate), poly U (Miles, ammonium salt), poly C (Miles, potassium salt, s = 4.27), poly A (Miles, potassium salt, s = 13.1), and poly X (Miles, s = 6.22), each at concentrations of 20 mg per ml, were thermally denatured either with or without formaldehyde (1% final concentration). All preparations were then batch-treated for 2 hr at 40 with Sephadex G-25 (preswollen in 0.15 N NaCi). Yeast RNA (Sigma) was further purified by chromatography on Sephadex G-200 with 0.1 M NH4HCO3, pH 7.8; the material in the first peak was precipitated with ethanol. When included, RNA denaturation with formaldehyde was performed prior to this chromatographic step. Chicken embryo RNA was prepared22 with substitution of bentonite for polyvinylsulfate. It was denatured and partly purified as outlined above. Commercial tRNA (General Biochemicals, yeast) was further purified by chromatography on DEAE-cellulose.23 The fraction eluted with 1 N NaCl-0.1 N Tris-HCl, pH 7.5, was rechromatographed on Sephadex G-100 which had been equilibrated with 1 N NaCl.24 Hydrolysis of 18.7 mg of this sample in 1 ml TBS with 16.5 jsg pancreatic ribonuclease was carried out for 18 hr at 37°. The reaction was terminated by freezing and was subsequently analyzed by thin-layer chromatography.25 The distribution of products indicated partial hydrolysis into high-molecular-weight oligonucleotides. Parallel chromatographic experiments using a DEAE-cellulose column (0.1 M Tris-HCl, pH 7.52, with a linear gradient of NaCl from 0.1-1.0 M) confirmed this observation. Results.-The heterologous precipitin reactions obtained with globulin fractions and the RSA-nucleoside conjugates are shown in Figure 2. Precipitation with these hapten conjugates demonstrated that specific antibodies were formed. Comparable precipitin reactions between anti-Q globulins and Q-HSA were also found (Fig. 3). Here too, no precipitation was obtained with HSA at the same concentrations. The specificity of these reactions was further investigated by hapten inhibition experiments. Sufficient antigen was added to precipitate the maximum amount of antibody, as determined from the precipitin curves (Figs. 2 and 3). In the anti-U, U-RSA system (Table 1), uridine was a much better inhibitor than pseudouridine, dihydrouridine, or other haptens tested. Since uracil and ribose were each poor in- hibitors, the entire nucleoside appears to be involved in this antigenic determinant, as was reported earlier.6 Anti-+-globulins were inhibited almost equally by uridine Downloaded by guest on September 24, 2021 VOL. 57, 1967 BIOCHEMISTRY: KAROL AND TANENBAUM7715

MICROGRAMS ANTIGEN PROTEIN ADDED

O 10 50 100 I ' .60

50Co,50 ~~~~~~~~~ANTI- U +URSA

* .40 _ i 50 .40 20

E A 0 a t * * ANTI- + YS

.10

0 2 4 6 8 10 12 14 16 i8 20 MICROGRAMS ANTIGEN NITROGEN ADDED FIG. 2.-Precipitin reactions between globulin fractions from anti- nucleoside sera and various heterologous antigens. *, anti-U (1:4 dilution); *, anti-+/ (1:5 dilution); A, anti-red. U (1:5 dilution, previously absorbed with red. RSA). Corresponding open symbols represent reactions with RSA. Globulin dilutions (200 ;I) were brought to final reaction volumes of 0.5 ml with TBS. After 5 days at 40, the specific precipitates were washed twice with saline. The concentrations of U-RSA, 4-RSA, and RSA were determined by the Markham modification of the Kjeldahl procedure.'6 Other antigen concentrations were obtained by weight. and by pseudouridine, and less by dihydrouridine, dihydrouracil, and uracil. Ap- parently, the specificity of antibody populations to this determinant is not as re- stricted as is the case with anti-U sera. Anti-red. U-globulins were inhibited almost to the same degree by dihydrouridine, uridine, and cytidine, and somewhat less by pseudouridine (Table 1). The hapten inhibition pattern of the anti-Q globulins is shown in Table 2. Q- directed antibodies possess a high degree of specificity towards this moiety. Equiv- alent inhibitory activity was noted only with 500-fold higher concentrations of pyrimidine nucleosides. None of the Q-haptens caused inhibition of precipitation in a BSA, anti-BSA system. The high inhibitory activity of Q-tyrosine is in keeping with the presence of monoazotyrosine substitution in Q-HSA. Confirmation of the foregoing antibody specificities was obtained in gel-diffusion studies (to be presented elsewhere). The results of gel diffusions of these antisera against polyribonucleotides are summarized in Table 3. Each of the hapten-specific globulin fractions precipitated with poly X, whereas no precipitation occurred with poly A. Thus, the interaction of these immunoglobulins with polyribonucleotides is a constitutive manifestation of their specificity for the diketopyrimidine moiety. Failure to observe lines of precipitation of poly U with several anti-U preparations was not due to the presence of ribonuclease2" in these globulins. Additional evidence for the cytidine reactivity (Table 1) of anti-red. U sera came from the observation that these antibodies precipitated in gel with poly C. Failure of an anti-BSA globulin solution of similar nitrogen content to precipitate with any of the poly- Downloaded by guest on September 24, 2021 716 BIOCHEMISTRY: KAROL AND TANENBAUM PROC. N. A. S.

E TOTAL O.D. 285mp .04 E

6.3 ~~~~~~~~~~~~~~~~~~~~~.15a O.D.285mp(ANTlBODY PPTD.) 6

1.2 0 20 40 6 0)02

.1 ~~~~~~~~~~~~~~~~~~~.05

0 20 40 60 80 100 120 MICROGRAMS ANTIGEN ADDED FIG. 3.-Precipitin reaction between the globulin fraction (200 .l, undiluted) from an anti-Q serum and Q-HSA. The washed precipitates were dissolved in 0.5 ml 0.1 N NaOH for the following measurements: X, OD at 360 mu; o, OD at 285 mu; 0, OD at 285 mu after deduction of antigen contribution at this wavelength.

nucleotides eliminated the possibility that these gel precipitations were a function of hyperimmune sera. As with previously studied antisera to synthetic conjugates,25 globulin fractions from antisera of each of the four specificities described here precipitate with formaldehyde-denatured calf thymus DNA. The ability of several of these antisera to fix complement in reacting with RNA is depicted in Figure 4. Treatment of the yeast RNA with pronase (enzyme to substrate ratio 1: 60) for six hours at 370, followed by boiling for ten minutes, did not affect its C'-fixation with anti-red. U globulin, thus eliminating the possibility of spurious protein interactions. Although anti-U and anti-red. U immunoglobulins were each noted to fix some 30 per cent of the added complement with heat-denatured calf thymus DNA, ultracentrifugal density gradi- TABLE 1 INHIBITION OF QUANTITATIVE PRECIPITIN REACTIONS Globulin Preparation from Anti-U--- - AnAnti-* - Anti-Red. U- - Inhibition Inhibition Inhibition Ilapten ;&Moles (%) ;Moles (%) -,Moles (%) ridine 1.5 29 5.1 31 12.0 30 5.0 62 Pseudouridine 5.0 12 5.0 36 12.0 20 10.0 16 Dihydrouridine 5.0 22 5.0 14 11.9 34 9.9 26 11.9 20 C Widine 10.0 14 10.0 15 12.0 33 TLymldmne 10.0 15 9.9 1 12.0 8 Uracil 4.6 9 5.5 11 5.6 8 Dihydrouracil 5.5 13 5.5 5 Ribose 10.0 2 10.0 0 12.0 15 Total reaction volumes were 0.50 itil containing 0.20 ml immune globulin dilutions and the amount of antigen required for maximum precipitation. Downloaded by guest on September 24, 2021 VOL. 57, 1967 BIOCHEMISTRY: KAROL AND TANENBAUM 717

TABLE 2 HAPTEN INHIBITION OF Q-HSA PRECIPITATION WITH ANTI-Q GLOBULIN Hapten pMoles Inhibition (%) Q-tyrosine 0.025 26 Q-arbutin 0.031 11 Q-barbituric acid 0.062 15 Q-sulfosalicylic acid 0.40 34 Uracil 0.68 3 5.70 2 Thymine 0.51 3 4.27 10 Adenine 0.52 0 3.32 0 Guanine 0.50 0 1.44 0 Uridine 13.2 16 11.1 13 Cytidine 13.2 25 Thymidine 13.3 23 Pseudouridine 13.1 0 4-OH quinazoline 16.0 0 Total reaction volumes 0.51 ml, including 0.20 ml anti-Q globulin, and the amounts of antigen required to give maximum precipitation (Fig. 3). TBS buffer containing 20% acetone was used for more insoluble Q-haptens; corresponding control tubes also contained organic solvent. Antibody precipitated was calculated from solving three simultaneous equations involving total absorbance at 360, 285, and 305 my& and the EM values of antigen, antibody, and haptens at these wavelengths. ent analysis showed that no more than 3 per cent DNA was present in the chick embryo (C.E.) RNA sample which was used. The precipitation of formaldehyde-denatured yeast and chick embryo RNA's with an anti-Q globulin fraction is presented in Figure 5. A globulin preparation made from the preimmunization serum of this rabbit, or anti-BSA globulins, did not react under identical conditions. Of 390 jug chick embryo RNA added, two methods of analysis (legend, Fig. 5) gave 13 Ag RNA in the immune precipitate. Precipitation of formaldehyde-denatured yeast RNA by anti-Q, anti-U, and anti-red. U globulin fractions was also obtained in suitably controlled gel diffusion experiments. These reactions were shown to be inhibited by the presence of enzymatic or alkaline hydrol- yzates of RNA. Yeast tRNA failed to precipitate under these conditions. How- ever, it was demonstrated that ribonuclease-treated tRNA precipitated in gel with each of the four specific immunoglobulins described here. Discussion.-Various methods have been used to induce anti-RNA antibodies. Immunization with bacterial ribosomes elicited antibodies which precipitated with RNA, and with polyribonucleotides.28 The specificity of such antibodies appears to be directed toward the polyribose-phosphate backbone of RNA.29 Antibodies to AMP-BSA5 which precipitate with various DNA's and with poly A30 are inhibited by the presence of RNA in these reactions.A0 31 Complexes of RNA's with methylated BSA (mBSA) have successfully induced RNA-reactive antibodies.32 33 These re- TABLE 3 GEL PRECIPITATION OF POLYRIBONUCLEOTIDES BY GLOBULIN FRACTIONS FROM ANTISERA Poly U Poly C Poly X Poly A Anti-U - + Anti-# + + Anti-red. U + + + Anti-Q + + Anti-BSA - - - Downloaded by guest on September 24, 2021 718 BIOCHEMISTRY: KAROL AND TANENBAUM PROC. N. A. S.

100

sox FIG. 4.-Complement fixation reactions of an anti-U serum, 60 / and of an anti-red. U globulin fraction (absorbed with red. La 4% / RSA), with variously treated RNA preparations. Preimmuni- L 20 ', cl zation serum (1:700) from the same anti-U rabbit fixed a maximum of 18% C' with unheated yeast RNA, and 12% C' with 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 heated yeast RNA. At 1:50), MICROGRAMS OF RNA preimmunization serum from the * ANTI-U(1 700)+NAT.YEAST RNA 0 ANTI-U(1:700)+DEN.YEAST RNA anti-red. U rabbit failed to fix & ANTI-RED.U1:700)*DEN.YEAST RNA X ANTI-U(111200)+CHO-DEN.YEAST RNA C' with heated yeast RNA. A ANTI-RED.U(1:700)+NAT.YEAST RNA U ANTI-RED.U(1I700)+DEN.C.E. RNA 0 ANTI-RED U(1:700)* NAT. C.E. RNA

ports, together with the data given here and by Sela et al.,6 indicate that no funda- mental barrier exists to the formation of RNA-reactive antibodies. Natural com- ponents of RNA are not even required as parts of the immunogens, as exemplified by the case of the quinazolinedione antigen. The necessity for removal of serum ribonucleases in order to observe reaction of antisera with RNA or with polyribonucleotides has been documented.6 In our ex- perience, this step has not been found obligatory for demonstration of RNA cross- reactions. Furthermore, by preparation of the sodium-sulfate-precipitated globulin fractions, up to 90 per cent of antiserum ribonuclease activity was removed, and it was found that no more than 10-2 micrograms of RNase was present per ml of the reaction mixtures. Reactivity of tRNA with the specific antibodies was actually facilitated by prior hydrolysis with ribonuclease. This treatment may have exposed additional sites for antibody cross-reactions without forming products small enough to cause hapten inhibition. The apparent cytidine specificity of antisera to red. U may be related to re- ports'4 35 that ultraviolet irradiation of uridine gave a product which coded as cytidine. On the other hand, with irradiation at 500, Rottman and Cerutti36 could not find cytidine coding in a reduced poly U. In the synthesis of red. U antigens (am- bient temperature), the decreased ultraviolet absorbance, and the alkaline cleavage results indicated 70 per cent C5-C6 ring substitution. It can be assumed that dihydrouridine, the water adduct, dimers, and other photochemical side products such as amino acid-pyrimidine ligands, account for the properties of red. U albumins. This presumptive composition was reflected upon analysis of the specificity of anti-red. U sera (Table 1). The conformation of RNA is probably altered from the "native" state under the reaction conditions necessary to evince precipitation or C'-fixation with specific antibodies. Thus, variations in the shapes of precipitin curves of antiribosomal antibodies with ribopolymers have been observed following changes in ionic strength of the medium.37 However, under carefully controlled, selected conditions, the base- specific RNA-reactive antibodies described here may prove useful as an immunologic tool for following changes in RNA conformation, as already indicated in the work of Seaman et al."3 with antisera to mBSA-ribopolynier coml)lexes. Conversely, the Downloaded by guest on September 24, 2021 VOL. 57, 1967 BIOCHEMISTRY: KAROL AND TANENBAUM 719

I I I I I x .12 .60

CH2O-DEN. YEAST RNA < .10o ° (LEFT SCALE)/1 .505

O4.4

e .08.06..30.40

E 0 aH2tDEN.C.aE. RNA maotn04 in2(RIGHT SCALE) i 0

.02X .~~~~~~~~~~~~~~~~~~10

0 100 200 300 400 500

MICROGRAMS RNA ADDED

FiG. 5.-Precipitin reaction of an anti-Q globulin (125 th, undiluted), with formaldehyde denatured RNA's. Final reaction volumes were 0.525 ml. Re- sults of duplicate determinations are plotted. Specific precipitates with chick embryo RNA were washed, drained, and extracted with 1.0 ml of 1 N HCl4 for 20 min at 70g.26 Residual protein was analyzed by the biuret reaction. The absorbance of the supernatants was measured at 260 and 280 ma.". The

orcinol test27 was then performed on this extract. OD values at 660 mji were converted intojS g RNA from a standard curve obtained with chick embryo RNA.

actual combination of antibodies with RNA may cause configurational changes, or

may mask potentiating regions, even in the absence of overt immunochemical mani- festations. It follows that another application for RNA-reactive antibodies is in

their possible effect on RNA translation. Studies with the specific antisera de-

scribed in th is report upon RNA coding for in vitro protein synthesis are in progress.

The authors are indebted to Dr. Sam M. Beiser for his advice on many aspects of the immuno-

chemical techniques which were employed in this investigation. They also thank Dr. H. S. Rosen- kranz for chick embryo RNA and for ultracentrifugal analyses.

* Predoctoral trainee, grant 5 Fl-GM-22807, from the National Institutes of Health. This

report is taken from a dissertation to be submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Columbia University. 1Rosenkranz, H. S., B. F. Erlanger, S. W. Tanenbaum, and S. M. Beiser, Science, 145, 282 (1964). 2 Butler, V. P., S. M. Beiser, B. F. Erlanger, S. W. Tanenbaum, S. Cohen, and A. Bendich, these PROCEEDINGS, 48, 1597 (1962). Tanenbaum, S. W., and S. M. Beiser, these PROCEEDINGS, 49, 662 (1963). 4Beiser, S. M., S. W. Tanenbaum, and B. F. Erlanger, Nature, 203, 1381 (1964). Erlanger, B. F., and S. M. Beiser, these PROCEEDINGS, 52, 68 (1964). 6 Sela, M., H. Ungar-Waron, and Y. Schecter, these PROCEEDINGS, 52, 285 (1964); Federation Proc.. 24, 1438 (1965). Downloaded by guest on September 24, 2021 720 BIOCHEMISTRY: KAROL AND TANENBAUM PROC. N. A. S.

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