The importance of the lysyl residue in goat anti-dinitrophenyl antibodies modified by site-directed reagents

lmmunochemistry, 1973, Vol. 10, pp. 337-340. Pergamon Press.

Printed in Great Britain

T H E I M P O R T A N C E OF T H E L Y S Y L R E S I D U E IN GOAT ANTI-DINITROPHENYL ANTIBODIES M O D I F I E D BY S I T E - D I R E C T E D R E A G E N T S Y A C O B W E I N S T E I N , S H M U E L S H A L T I E L , D A V I D G I V O L and PAUL H. STRAUSBAUCH* Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel (First received 27 September 1972; in revised form 2 November 1972)

A series of affinity labeling bromoacetyl derivatives of the dinitrophenyl hapten has been shown to react with purified goat anti-dinitrophenyl antibody preparations at tyrosyl and lysyl residues. By means of the reversible amino group modifying reagent citraconic anhydride, it was possible to show that the lysyl residue modified by the affinity labeling reagents was not essential to antigen binding. Hence this reactive lysyl residue is probably situated outside of, but close to the hapten combining site. Additional experiments revealed the existence of two distinct types of goat IgG, a population which reacts with the affinity labeling reagents exclusively through tyrosyl residues, and one which reacts exclusively through lysyl residues. There does not appear to be a third population of antibodies which reacts both with tyrosyl and lysyl residues. Abstract-

INTRODUCTION

A series of bromoacetyl derivatives of the dinitrophenyl (DNP) hapten has recently been used to study the combining sites of anti-DNP antibodies (Weinstein et al., 1969). These reagents have demonstrated the importance of tyrosine in the antibody combining site as shown in previous work (Grossberg et al., 1962; Wofsy and Singer, 1962). I n Contrast to these experiments, a particular lysyl residue was modified when the bromoacetyl derivatives of the D N P - h a p t e n were reacted with anti-DNP antibodies isolated from immune goat serum (Strausbauch et al., 1971). in fact the antibodies isolated from one particular goat reacted predominantly through lysyl residues. The question arose whether or not this lysyl residue was important for antigen binding in the antibodies isolated from this individual, i.e. is the lysyl residue modified by this series of reagents a 'contact' residue within the combining site. Previous work has implicated lysyl residues as being in the antigen binding site of a limited number of antibody preparations, elicited in response to injection of very acidic immunogens, but has failed to demonstrate the importance of lysyl residues in antibodies directed towards a neutral hapten like the DNP-group (Freedman et al., 1968; Grossberg and Pressman, 1963; Joniau et al., 1970, Wofsy and Singer, 1963). The present communication reports: (l) An investigation of the importance of lysyl residues in goat *Postdoctoral fellow of the American Cancer Society. Present address: Department of Immunology, University of Manitoba, Winnipeg, Canada_

anti-DNP-antibodies, especially that lysyl residue which was modified by the bromoacetyl sitedirected reagents. (2) The demonstration of at least two different types of antibody combining sites, one with a tyrosine near or at the hapten binding site, and one with a lysine probably near the hapten binding site. This study takes advantage of reversible amino modification by citraconic (methylmaleic) anhydride (Dixon and Perham, 1968) for assessing the importance of lysyl residues in these antibodies. MATERIALS AND METHODS

Preparation and characterization of antibodies. Anti-DNP specific antibodies were produced in goats injected with DNP-keyhole limpet hemocyanine and isolated from their sera on a DNP-rabbit serum albuminSepharose immunoadsorbent, prepared as described by Givol et al. (1970). Details of the injection schedule can be found in Strausbauch et al. (1971). Two preparations of goat IgG were used in these studies: one from goat 8, isolated from a single bleeding taken 8 months "after the initial injection, and one from goat 44, isolated from a single bleeding taken 7 months after the initial injection. Site-directed reagents label the goat 8 preparation mainly at tyrosyl residues (70-90 per cent), while these same reagents label the goat 44 preparation predominantly at lysyl residues (60-85 per cent) (Strausbauch et al., 1971). These labeling patterns are characteristic for the two individual animals and did not vary with subsequent immunization, or time of bleeding. The mol. wt of goat lgG was taken to be 144,000 with an absorbance index, A ~ of 13-0 (Givol and Hurwitz, 1969). The degree of hapten binding was determined by fluorescence quenching titrations according to Eisen and Siskind (1964). Reversible modification of aminogroups. Goat igG

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YACOB WEINSTEIN et al.

was modified by reaction with citraconic anhydride (BDH) under conditions similar to those described (Dixon and Perham, 1968; Pull, 1970). Citraconyl groups were removed by 24 hr dialysis against 0.1 N Na acetate, pH 3-5, 20°C. Extent of amino group modification was determined by the trinitropbenylsulfonate method of Habeeb (1966). Simultaneous controls were run with bovine serum albumin, ovalbumin and unmodified goat IgO as standards. The molar absorption of trinitrophenyllysine was found to be 1.1 x 104 at 335 nm by comparison of the intensity of color produced in this reaction with the amino acid analyses of the standards. This value is in good agreement with those values (0.99-1.24× 10') reported in the literature (Habeeb,

1966). Affinity labeling reagents and conditions of reactions. The radioactive affinity labeling reagents N-bromoacetylN'-DNP-ethylened/amine (BADE), and N°-bromaocetyb N'-DNP-L-lysine (BADL) were sytbesized as described by Weinstein et al. (1969). Affinity labeling of purified immunoglobulins was carried out in a reaction mixture composed of antibody (I x 10-6M), the p'C) affinity labeling reagent, BADE or BADL (4x 10-eM), in 0.1 M NaHCOa, pH 9.0. The reaction was run at 37°(: and the extent of covalent labeling was determined by incorporation of radioactivity into trichloracetic acid precipitates (Weinstein et al., 1969). Relative degree of labeling at tyrosyl or lysyi residues was analyzed on acid hydrolyzates (6N HCI, 108°C, 24 In') of the modified protein. The resulting (14C)-carboxymethyl amino acids were separated and identified by paper electrophoresis (Strausbauch et al., 197 i). Reagents. DNP-protein conjugates were prepared as described by Eisen et aL (1953). DNP--ovalbumin coupled to Sepharose 4B immunoadsorbent was prepared according to Porath et al. (1967) as described by Givol et al. (1970). The reagents used in this study were of the highest grade commercially available and used without further purification.

DNP-aminocaproate). Although this is the usual result to be expected with most antibody preparations (Grossberg and Pressman, 1963; Wofsy and Singer, 1963), it was particularly significant that lysyl residues did not appear to be essential for hapten binding in that antibody preparation (goat 44 antibodies) in which a lysyl residue was modified by site-directed affinity labeling reagents (Strausbauch et al., 1971). The above experiment indicated that lysyl residues in lgG from goats 8 and 44 are not important for hapten binding. However, since the lysyl residues in goat IgG modified by the affinity labeling reagents may be situated at some distance from the site of D N P binding (5-10 A, Strausbauch et al., 1971), there is the possibility that modification of this lysine will have no effect on binding of a small hapten, but will have a marked effect on binding of the complete antigen ( D N P - p r o t e i n conjugate) through steric hinderence. To test this hypothesis, the ability of the modified immunoglobulin to bind a large antigen, D N P - o v a l b u m i n , was tested. Solutions of native and citraconylated anti-DNP antibodies were incubated with D N P - o v a l b u m i n Sepharose immunoadsorbent. The degree of antibody binding to the insolubilized antigen was then assesed by determining the amount of protein remaining in the supernatant after centrifugation I

I

I

I

I

!

90 ~

RESULTS Citraconylation o f antibody amino groups. Standard preparations of citraconylated goat 8 and 44 antibodies were made in the following manner. To a solution of purified goat IgG (5mg/ ml) in 0 - 3 M N a H C O s , pH 8.5 (15ml total) was added 0.05 ml of citraconic anhydride in small portions over a period of 30 rain at 24°C. During this time the mixture was constantly stirred and maintained at pH 8.5 by addition of 5 N N a O H . At the end of this time, 200 mg of glycine was added and the solution extensively dialyzed a ~ i n s t 0"15 M NaCI-0.02 M N a phosphate, pH 7.2. Properties o f the citraconylated antibody preparations. The extent of lysine modification of the anti-DNP antibodies from goats 8 and 44 was 76 and 82 per cent respectively, as determined by trinitrophenylsulfonate titration. In spite of the fact that the lysyl residues are almost completely reacted, these antibody preparations have not lost their ability to bind hapten as shown by their fluorescence quenching with D N P iysine (Fig. 1), (a similar result was obtained with

I

8O

7O ~60

40

O I I

I 2

I 3

I 4

~ I

I

5

6

N'DNP Lys.(Moles x 10"9) Fig. I. Fluorescence quenching of native and citraconylated goat anti-DNP antibodies by addition of c-DNP-lysine. Each titration was performed with 1.62x10-~mole of antibody in 0.15M NaCI-0.02 Na phosphate, pH 7.2, 25° (2.5nd total volume). The solutions were activated at 280nm and fluorescence determined at 335 nm. All data is corrected for dilution. ( - E l - ) native goat 44 anti DNP; ( - 1 7 - ) citraconylated goat 44 anti DNP: ( - O - ) native goat 8 anti DNP: ( - © - ) citraconylated goat anti DNP.

Lysyl Residues Modified by Site-Directed Reagents (Table 1). Citraconylation of lysyl residues has no effect on the ability of either goat 8 or 44 antibody preparations to bind a large antigen molecule. The two types of antibodies have not been compared by precipitin tests, so they may still differ by this criteria. Table 1. Binding of native and citraconylated anti-DNP antibody to DN P-protein immunoadsorbent Preparation '~

Total protein b Total protein %lgG applied eluted bound

Native goat 8 IgG

2.28 mg

0-42 mg

72%

Citraconylated goat 8 IgG

3.70 mg

0.75 mg

80%

Native goat 44 IgG

1.32 mg

0.21 mg

84%

Citraconylated goat 44 IgG

5.22 mg

1.32 mg

75%

~Preparations are those described in text. bTo 2 ml of the antibody solutions in 0.15 M NaCI0.02M Na phosphate, pH 7.2, was added 1 ml of a 50% suspension of DN P--ovalbumincoupled to Sepharose 4B immunoabsorbent. These mixtures were stirred for 2 hr at 37°C and I hr at 24°C, followed by centrifugation. Optical density was determined on the supernatant and three subsequent washes in the same buffer. Values are reported as total optical density at 280 nm applied and eluted from the immunoadsorbent.

Affinity labeling studies on citraconylated immunoglobuHns. Since citraconic anhydride does not completely react with all the lysyl residues in the lgG molecule (15-25 per cent remain), it was essential to show that the lysyl residue which reacted with the affinity labeling reagents was modified by the citraconylation procedure. If this lysyl residue was not modified by citraconic anhydride, but was present in the antigen binding site, it would explain why modification of a majority of the lysyl groups would have no effect on antigen binding. Native and citraconylated goat igG were reacted with either B A D E (goat 8 IgG) or BADL (goat 44 IgG). These reagents were chosen for maximal reaction with the respective immunoglobulins (Strausbauch et al., 1971). Samples were analyzed for the extent of reaction and for the relative incorporation of label into tyrosyl and lysyl residues (Table 2). Modification of lysyl residues by citraconylation results in almost complete blockage of affinity labeling at lysine with neglible effect on labeling at tyrosine. Removal of the blocking groups by dialysis against Na acetate buffer, pH 3-5, followed by affinity labeling with either BADE or BADL showed full reconstitution of reaction at lysyl residues.

339

Different populations of affinity labeled antibodies. An additional point of interest in these experiments was the unequivocal demonstration of at least two different populations of goat antibodies: a type which reacts with affinity labeling reagents through lysyl residues (the major species in goat 44), and a type which reacts through tyrosyl residues (the major species in goat 8). This was accomplished by a double labeling procedure involving the citraconylated antibody. Citraconylated lgG from goats 8 and 44 were reacted with non-radioactive BADE, followed by dialysis against 0-1 M Na acetate, pH 3.5, to remove the attatched citraconyl groups. These preparations were then concentrated and dialyzed against 0.15M N a C I - 0 - 0 2 M N a phosphate, pH 7-2. The cold B A D E labeled decitraconylated immunoglobulin was then reacted with either radioactive BADE (goat 8) or BADL (goat 44) under standard conditions. Analysis of the radioactivity incorporated revealed insignificant reaction of the radioactive reagents with tyrosyl residues and almost unimpaired reaction at lysyl residues (Table 2). Modification of lysyl residues with citraconic anhydride did not increase the amount of reaction at tyrosine, and likewise, affinity labeling of the tyrosyl residue did not decrease reaction at lysine. This result indicates that there is a population of antibody molecules which reacts with bromoacetyl affinity labeling Table 2. Covalent reaction of citraconylated goat antiDNP antibodies with affinity labeling reagents

Unmodified protein a Goat 8

TYR a LYSa 1.30 0.14

Goat 44 0.15

0.52

Citraconylated proteina-b

Protein modified with cold BADEa-b.¢

TYR O LYSa TYR ~ LYSd 0.83 0.03 0.06 0.18 0-10

0.11

0.02

0-44

~Goat anti-DNP antibodies, and modified antibody preparations were reacted with BADE (goat 8) or BADL (goat 44) under the standard conditions for 20 hr. bCitraconylated preparations of purified anti-DNP antibodies were prepared as described in text. ~'Citraconylated goat anti-DNP antibodies were reacted with non-radioactive BADE under standard conditions of 20hr. Citraconyl blocking groups were then removed by dialysis against 0.1 M Na acetate, pH 3.5, for 24 hr at 24°C, followed by extensive dialysis against 0.15M NaCI-0.02M Na phospates, pH 7.2. The BADE labeled, unblocked immunoglobulin was then reacted with either radioactive BADE (goat 8) or BADL (goat 44) under the usual conditions. dExtent of modification is given as number of moles of radioactive reagent incorporated into either tyrosine or lysine per 2 mole of hapten combining sites, or 1 mole of antibody.

340

YACOB WEINSTEIN et al.

reagents exclusively through tyrosyl residues, and one which reacts exclusively through lysyl residues. Goat 8 produces antibodies mostly of the former type while goat 44 the latter. There does not appear to be a third population of antibodies with both a reactive tyrosyl and lysyl residue in the same combining site. DISCUSSION The principal conclusion of this study is that the lysyl residue in goat anti D N P which is modified by bromoacetyl affinity labeling reagents is not essential for hapten or antigen binding. This is a rather interesting finding since the dimensions of the bromoacetyl reagents (17 A for B A D L , the largest used) are such that they should be able to be accomodated within the hapten binding site (measured experimentally to be 2 5 - 3 4 A ; Kabat, 1966). As a result, one would expect any residue modified by these sitedirected reagents to be situated in the combining site. There are two possible explanations for this result. One is that this particular lysine is within the combining site, that it is not essential for hapten binding, and that it is not a 'contact residue', since attatchment of a bulky citraconyl group causes no steric hindrance of hapten or antigen binding. The second possibility is that this lysyl residue is not within the combining site, but only on the periphery of it. This is a definite possibility since the values given for the size of the combining site are taken from the dimensions of the test haptens in thier most extended form (Kabat, 1966). Since most of these determinations were made with biopolymers, polysaccharides or synthetic polypeptides, which can assume multiple conformations, the dimensions of the bound hapten may be much less than the extended dimensions. There is no possibility that these bromoacetyl reagents are not site-directed, and have reacted at a residue removed from the vicinity of the combining site. This is shown by the fact that (1) these reagents do not react with normal nonimmune IgG (Strausbauch et al., 1971; Weinstein et al., 1969); (2) the reaction is blocked by addition of hapten; (3) covalent attatchment of B A D E or B A D L to the immunoglobulin results in fluorescence quenching of tryptophan residues similar to that observed with addition of free hapten, a result which would not occur if the DNP-group of the labeling reagent was not situated in the hapten combining site (Eisen and Sisking, 1964; Strausbauch et al., 197 I). The second important finding of this report is the demonstration of two distinct populations of antibody. Any one molecule of IgG reacts with the affanity labeling reagent at either lysine or

tyrosine. There is no group of antibody molecules which have both a reactive lysine and a reactive tyrosine. Previous studies have shown the importance of tyrosyl residues and have suggested that there are essential tyrosyl residues present in the combining site of all antibodies (Grossberg et aL, 1962; Wofsy et ai., 1962). These results do not conflict with this conclusion, but point out that in certain antibody preparations (such as from goat 44) these essential tyrosyl residues do not react with affinity labeling reagents although other residues in the vicinity will react with them. Furthermore, prevention of affinity labeling at lysine by blockage with citraconic anhydride does not increase the affinity labeling at the tyrosyl residue. Thus failure to react at tyrosine in certain antibody preparations is not due to the presence of an overly reactive lysyl residue which will prevent reaction at other residues, in like manner, depletion of the tyrosine reactive population of immunoglobulin has no effect on the amount of labeling at lysine. This study demonstrates the utility of citraconylation in the study of antibody combining sites. Although citraconylation changes the charge it has not changed the hydrophilic nature of the lysine which may contribute to the stability of the probable hydrophobic site. The simplicity of reaction and ease of blocking group removal recommends it to the study of protein molecules. REFERENCES

Dixon H. B. F. and Perham R. N. (1968) Biochem. J. 109, 312. Eisen H. N., Belman S. and Carsten M. E. J. (1953) J. Am. chem. Soc. 75, 4583. Eisen H. N. and Siskind G. W. (1964) Biochemistry 3, 996. Freedman M. H., Grossberg A. L. and Pressman D. (1968) lmmunochemistry 5, 367. Givol D. and Hurwitz E. (1969) Biochem. J. 115, 37 I. Givol D., Weinstein Y., Gorecki M. and Wilchek M. (1970) Biochem. biophys. Res. Commun. 38, 825. Grossberg A. L., Radzimski G. and Pressman D. (1962) Biochemistry 1, 39 I. Grossberg A. L. and Pressman D. (1963) Biochemistry 2, 90. Habeeb A. F. S. A. (1966)Analyt. Biochem. 14, 328. Joniau M., Grossberg A. L. and Pressman D. (1970) Immunochemistry 7, 755. Kabat E. A. (1966)J. Immun. 97, I. Porath J., Axen R. and Ernback S., (1967) Nature, Lond. 215, 1491. Pull 1. (1970) Biochem. J. 119, 377. Strausbauch P. H., Weinstein Y., Wilchek M., Shaltiel S. and Givol D. ( i 97 I) Biochemistry 10, 4342. Weinstein Y., Wilchek M. and Givol D. (1969) Biochem. biophys. Res. Commun. 35,694. Wofsy L. and Singer S. J. (1962) Biochemistry 1, 1031. Wofsy L. and Singer S. J. (1963) Biochemistry 2, 104.