Biochimica et Biophysica Acta, 400 (1975) 423-432

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37097 E N Z Y M I C A N D I M M U N O C H E M I C A L PROPERTIES OF LYSOZYME IX. C O N F O R M A T I O N A N D I M M U N O C H E M I S T R Y O F DERIVATIVES S U C C I N Y L A T E D AT C E R T A I N LYSINE RESIDUES

CHING-LI LEE a, M. Z. ATASSIa'* and A. F. S. A. HABEEBb aDepartment of Chemistry, Wayne State University, Detroit, Mich. 48202 and bDivision of Clinical Immunology and Rheumatology, Departments of Medicine, Microbiology and Biochemistry, University of Alabama School of Medicine, Birmingham, Ala. 35233 (U.S.A.)

(Received January 20th, 1975)

SUMMARY Succinylation of lysozyme in the presence of 7 molar excess of [1,4-14C2]succinic anhydride gave a reaction product which showed at least six components by disc electrophoresis. Chromatography on CM-cellulose enabled the isolation of six homogeneous derivatives. The derivatives were succinylated at the following locations: derivative I, lysines-1 (a- and e-NH2), -13, -97 and -116 and the OH group at position 43 (or 36 or 40); derivative lI, lysines-1 (a- and e-NH2), -13, -96, -116; derivative III, lysines-1 (a- and e-NH2), -13, -97 and -116; derivative IV, lysines-1 (t~-NH2), -33, -96 and - 116; derivative V, lysines- 1 (tt-NH2), -33 and -96; derivative VI, lysines-33 and -116. Conformational changes were detectable in derivative I by ORD and CD measurements and by accessibility of the disulfide bonds to reduction. On the other hand, the other five succinyl derivatives showed no conformational changes by O R D and CD measurements. However, their disulfide bonds were slightly more accessible to reduction than lysozyme, with the increase being somewhat higher in derivatives I, II and III. Enzymic activity measurements showed that only derivative VI possessed some (10 %) enzymic activity. Immunochemical studies with antisera to lysozyme showed that the reactivity of each of the derivatives was lower than the homologous reaction. Correlation of the extent of decrease in immunochemical reaction with the locations of modification and with the results of conformational analysis, led to the conclusion that lysines 33, 96 and 116 are part of antigenic reactive regions in lysozyme. The modification results are also discussed in relation to the threedimensional structure of lysozyme in solution.

INTRODUCTION Determination of the antigenic structure of a native protein is a complex task that requires a strategy relying on a variety of approaches for appropriate solution * To whom correspondence should be addressed.

424 (see refs 1 and 2). The undertaking is even more difficult with disulfide-containing, proteolytically inaccessible (tight)proteins (for detailed discussion, see ref. 3). However, we have recently introduced a novel approach [3] which permitted a very appreciable delineation of the antigenic structures of lysozyme [3] and of bovine serum albumin [4]. Accordingly, determination of the complete antigenic structure of a tight protein, which has thus far been achieved only for myoglobin [I, 2, 5-9], has now become feasible. We have previously reported on the role in the antigenic structure of lysozyme and its conformation, of the tyrosine [10, 11], tryptophan [12], arginine [13] and dicarboxyl [14, 15] residues. In the present work lysozyme was succinylated and six succinylated lysozyme derivatives were isolated, characterized and their immunochemistry and conformation studied in detail. From the findings, it was possible to determine the involvement of lysines 33, 96 and 116 in the antigenic structure of native lysozyme. MATERIALS AND METHODS

Materials Hen egg-white lysozyme (three times crystallized) was from Sigma Chemical Co. and was homogeneous by starch gel, acrylamide gel and disc electrophoresis. Succinic anhydride was obtained from Aldrich Chemical Co. while the [1,4-14Cz-] labeled anhydride was from I.C.N. Isotope and Nuclear Division. Freeze-dried vials of Microcoecus lysodeikticus cells and trypsin-Tos-PheCH2C1 (trypsin pretreated with tosyl-phenylalanine chloromethyl ketone to eliminate chymotryptic activity), twice crystallized were purchased from Worthington Biochemical Corp. 2,4,6-Trinitrobenzenesulfonic acid was obtained from Eastman Chemical Co. and 5,5'-dithiobis(nitrobenzoic acid) was from Aldrich Chemical Co.

Reaction of lysozyme with suecinic anhydride and chromatography of the product To a magnetically stirred solution (150 ml) of lysozyme (3 g; 209.7/~mol) in 0.1 M sodium phosphate buffer at pH 8.0 was added succinic anhydride (150 mg; 1.499 mmol) in dioxane (4 ml) which also contained 50#Ci of [1,4-~4C2]succinic anhydride. Addition of the anhydride to protein was effected gradually over a period of 20 min and the pH was maintained at 8.0 on the pH star with 1 M NaOH. Reaction mixture was stirred further at room temperature for 30 min after which it was dialysed extensively against distilled water and freeze-dried. Chromatography of the reaction product was carried out on portions (0.40.5 g) of succinylated lysozyme on CM-cellulose columns (2 × 70 cm) which had been preequilibrated with 0.05 M sodium phosphate buffer (pH 4.5). The column was eluted with a linear pH gradient from pH 4.5 to 6.5. The mixing vessel contained 0.05 M sodium phosphate buffer, pH 4.5 (1 1) and the reservoir contained 0.05 M phosphate buffer at pH 6.5 (1 l). Chromatography was at 0 °C and the column was eluted at the rate of 20 ml per h. Effluent fractions (4.5 ml each) were read at 280 nm. Fractions belonging to one peak were combined, dialysed well against distilled water and freezedried. Material of a given peak from several chromatographic experiments was subjected to rechromatography once or twice on a similar column yielding completely homogeneous derivatives. The electrophoretically homogeneous components were dialysed extensively against distilled water and then freeze-dried.

425

Analytical methods Absorbance measurements were done with a Zeiss PMQII spectrophotometer. Disc electrophoresis was performed as described previously [12]. Extent of esterification of aliphatic OH groups was determined by the hydroxylamine-FeC13 procedure previously described [12]. The extent of amino group modification was determined by the trinitrobenzenesulfonic acid method [16, 17]. Counting of 14C label was performed with an Isocap/300 liquid scintillation system (Searle Analytic Inc.). Enzymic activity was determined in 0.06 M sodium phosphate buffer, containing 0.09 ~ NaC1 (pH 6.2), from the rate of lysis of M. lysodeikticus as previously described [10]. Concentrations of protein solutions were derived from their nitrogen contents which were determined by a micro-Kjeldahl procedure [18] and by using Nessler's reagent standardized with (NH4)2SO4. Three or more replicate analyses were usually performed and they varied i 0.5~o or less. The calculated nitrogen contents of lysozyme and derivatives are given in Table I. The values for the derivatives were calculated by taking into account the number of succinyl groups coupled to lysozyme. TABLE I MOLECULAR WEIGHTS, NITROGEN CONTENTS AND MEAN RESIDUE WEIGHTS OF LYSOZYME AND DERIVATIVES Values are calculated taking into account the number of succinyl groups incorporated into lysozyme. Protein

Mol. wt

Nitrogen Mean content residue (%) wt

Lysozyme Derivative I Derivative II Derivative III Derivative IV Derivative V Derivative VI

14 307 14 907 14 807 14 807 14 707 14 607 14 507

18.80 18.04 18.17 18.17 18.29 18.41 18.54

110.9 115.6 114.8 114.8 114.0 113.2 112.5

Reduction and S-carboxymethylation of the disulfide bonds in lysozyme and derivatives were performed as described elsewhere [19]. Procedures for tryptic hydrolysis [10] of these preparations and for peptide mapping [20] have also been reported. Peptide maps were stained with ninhydrin (0.2 ~ in ethanol) or with specific stains for various amino acids [21].

Evaluation of conformational changes Susceptibility of the disulfide bonds to reduction with 2-mercaptoethanol was used to monitor conformational changes [10, 23] using 5,5'-dithiobis(nitrobenzoic acid) [24]. Conformational changes were also determined by ORD and CD measurements of the protein solutions in water. The ORD and CD data were corrected for the refractive index dispersion of water and are, therefore, given here in [m'] and [0'], respectively. Experimental procedure and quantitative treatment of data were done as described elsewhere [25, 26]. The molecular weight and mean residue weight values

426 employed in the present calculations are summarized in Table I. For the calculation of b0 [27] a ;to of 212 nm was used.

Immunochemical methods Agar double diffusion, quantitative precipitin and absorption experiments were performed as described elsewhere [20]. Early-course antisera to lysozyme were raised in goats and in rabbits by the procedure previously reported in detail [28]. Antisera G9 and G10 were goat antisera and L1 and L2 were rabbit antisera, each against native lysozyme. Antisera of individual animals were kept and studied separately and stored in 5-8-ml portions at --40 °C. RESULTS

Succinylation of lysozyme and purification of the derivatives Reaction of lysozyme with succinic anhydride yielded a reaction product in which 65 % of the amino groups (i.e. 4.5 groups) were succinylated as determined by the trinitrobenzenesulfonic acid method. The reaction product, however, was heterogeneous by disc or acrylamide gel electrophoresis showing at least six distinct electrophoretic components. A reaction product of this type, containing several molecular species, is of course unsuitable for immunochemical and conformational studies. Column chromatography of the reaction product on CM-cellulose gave at least six components. Fig. 1 shows the chromatographic pattern of succinylated lysozyme. Each of the components needed rechromatography once or twice to obtain an electrophoretically homogeneous derivative.

Characterization of the derivatives The number of the amino groups modified, obtained by the trinitrobenzene-

1"4

=E 0 O0 O4

I'C IV

,-4 d

O.6[i

V

i F

I o

4'0

so

,20 ,60 TUBE NO ~-Sml)

260

' 240

Fig. 1. Chromatographic pattern of lysozyme succinylation product (400 mg) on a CM-cellulose column (2 x 70 cm). For details, see the text.

427 TABLE II TYPE AND LOCATION OF THE RESIDUES MODIFIED IN VARIOUS SUCCINYLATED LYSOZYME DERIVATIVES Derivative

NH2 Groups modified*

Nos of succinyl Locationsof the groups incor- modifications*** porated**

I

5.00

5.61

II III IV V VI

5.09 4.98 4.05 2.72 1.72

4.94 5.11 3.40 2.60 2.00

Lys-1 (a- and e-NH2), Lys-13, -97 and -116; OH group at position 43 (or 36 or 40) Lys-1 (a- and e-NH2) Lys-13, -96 and -116 Lys-1 (it- and e-NH2) Lys-13, -97 and -116 Lys-1 (ct-NH2), 33, 96, 116 Lys-1 (a-NH2), 33, 96 Lys-33, 116

* Determined by the trinitrobenzenesulfonic acid method [16, 17] and represent the average of four determinations. ** Determined from 14C label incorporated and represent the average of four replicate determinations. *** Determined by peptide mapping. For details, see the text. sulfonic acid reaction, in various derivatives is summarized in Table II. The table also gives the numbre of succinyl groups coupled to each derivative, as determined from the 14C label incorporated. Fig. 2 shows that electrophoretic mobility was directly related to the number of coupled succinyl groups. Except for derivative I, the number of succinyl groups coupled agreed well with the number of amino groups modified in each of the other derivatives. This agreement ruled out esterification of hydroxyl groups which can take place in acylation reactions [29, 30]. In derivative I, where there was one more succinyl group coupled than can be accounted for by the number of acylated amino groups, the results suggested succinylation of a hydroxyl group in the molecule. This was confirmed by the hydroxylamine-FeCl3 reaction which showed that 0.94 hydroxyl groups per mol of derivative I had been esterified. On the other hand, succinyl derivatives I I - V I possessed no succinylate hydroxyl groups. The locations of the modifications in each derivative were determined by peptide mapping of the tryptic hydrolysates of the S-carboxymethyl preparations from

w _.1 >-

I

z4 c~

.~'-"- IV L VI

"'~V

~ ~---MOBil_lTY(cm)----~• Fig. 2. A plot showing change in electrophoretic mobility, in polyacrylamide gel electrophoresis, with the number of succinyl groups incorporated. The Roman numerals refer to the corresponding chromatographic components of succinyl lysozyme (see Fig. 1).

428 lysozyme and its derivatives. In the maps of each derivative certain peptide spots disappeared (relative to the maps of lysozyme) and new spots appeared elsewhere in the maps. The spots that disappeared from the lysozyme maps in a given derivative were cut out from several lightly stained (0.05 ~ ninhydrin in ethanol) lysozyme maps, etuted with water, the eluates cleared by centrifugation, freeze-dried and their acid hydrolysates subjected to amino acid analysis. Comparison of the amino acid composition of the peptide with the known sequence of lysozyme [31] yielded its identity. The new spots that appeared in the derivatives, and which were also revealed by radioautography, were not cut out for elution and analysis because they overlapped with other lysozyme peptides on the maps. For brevity, the results of these extensive localization studies are summarized in a tabular form in Table II. With derivative 1, in addition to the acylated amino groups indicated in the table, one hydroxyl group was succinylated which was located on peptides 34-45. This peptide carries three hydroxyl groups located at serine-36, threonines-40 and -43 and we have not determined which of these three residues had been succinylated. However, since in the threedimensional structure [34], serine-36 and threonine-40 are quite buried while threonine-43 is exposed it is likely that threonine-43 was succinylated here.

Conformation of the derivatives The accessibility of the disulfide bonds to reduction in lysozyme and the derivatives is given in Table lII. In lysozyme, the disulfide bonds are inaccessible to the reducing agent. Conformational changes in the derivatives result in small but measur-

TABLE III ENZYMIC ACTIVITY AND DISULFIDE ACCESSIBILI'I'Y OF LYSOZYME AND DERIVATIVES Protein

Enzymic Disulfide reducibility* activity l h 3h

Lysozyme Derivative I Derivative II Derivative Ill Derivative IV Derivative V Derivative VI

100 0 0 0 0 0 10

0.043 0.16 0.16 0.15 0.10 0. l0 0.10

0.053 0.35 0.20 0.20 0.15 0.15 0.15

* Determined after reduction with mercaptoethanol for I and for 3 h, see the text.

able increases in reducibility of the disulfide bonds, with the largest change being observed in derivative 1. The reducibility of the disulfides in derivatives 1! and III, although lower than derivative I, was about four times higher than in native lysozyme. Conformational changes were also monitored by O R D and CD measurements which are summarized in Table IV. These revealed readily detectable changes only in derivative I, even though the b0 values of derivatives II and III may also be indicative of some small conformational changes.

429 TABLE IV ORD AND CD PARAMETERS OF LYSOZYME AND DERIVATIVES Determinations were carded out on the protein solutions in water. Protein Lysozyme Derivative Derivative Derivative Derivative Derivative Derivative

I II III IV V VI

ORD parameters [m'b.~a b 0

CD parameters [0']2z0 [0']z0a

--4420 --220 --3710 --182 --4328 --192 --4000 -- 195 --4320 --216 --4390 --220 --4440 --220

--7270 --9740 --5970 --8390 --7120 --9550 --7230 --9560 --7190 --9650 --7290 --9720 --7350 --9760

Enzymic activity of the derivatives The results o f enzymic activity measurements are summarized in Table III. Except for derivative VI, the derivatives entirely lost their lytic activity. Even in derivative VI, the lytic activity was only 10% relative to native lysozyme.

Immunochemistry of the derivatives With antisera to lysozyme, succinylated derivatives I, II and I I I showed no, or extremely faint, precipitin lines. O n the other h a n d derivatives IV, V and VI each gave a single, but weak, precipitin line showing no spurs or intersections. Quantitative precipitin analysis with antisera to lysozyme showed that succinyl lysozymes I, II and III, had small but appreciable immunochemical reactivity. The antigenic reactivities o f succinyl derivatives IV, V and VI were also suppressed relative to the h o m o l o g ous reaction. Fig. 3 shows an example o f the precipitin reaction o f lysozyme and

045r

0-'-'51 E 0'25

0'0

0

I'0

.'5"0 5! 0 ANTIGEN N (,ug)

i

Fig. 3. Quantitative precipitin reaction of lysozyme (©) and derivative VI (0) with antiserum G 9. Absorbance at 750 nm refers to determination of the protein in the immune precipitate by the FolinLowry method [46]. For reaction of all the succinylated lysozyme derivatives with several antisera. see Table V.

430 TABLE V QUANTITATIVE PRECIPITIN ANALYSIS OF SUCCINYLATED LYSOZYME DERIVATIVES WITH VARIOUS ANTISERA TO LYSOZYME Values are given in percent precipitation at equivalence relative to reaction of lysozyme with the respective antiserum. Results represent the average of three or more determinations whichvaried ± 1.1 or less. G9 and G10 are goat antisera and LI and L2 are rabbit antisera, each to native lysozyme. n.d., not determined. Protein

Lysozyme Derivative I Derivative II Derivative III Derivative IV Derivative V Derivative V1

Reaction with antiserum (~) G9

GI0

L1

L2

100 n.d. n.d. n.d. 42.5 53.7 64.0

100 12.5 24.5 24.4 42.8 56.4 62.4

100 18.6 20.7 24.6 41.1 55.5 60.7

100 15.3 20.4 29.4 40.0 57.4 68.5

derivative VI with one antiserum (G9) to lysozyme. The results of reaction of all the six derivatives with four different antisera are summarized in Table V. Values shown in Table V were also confirmed by absorption experiments in which antisera were absorbed with a given derivative and then the reactivity of the absorbed antiserum with native lysozyme was determined. DISCUSSION Acylation of lysozyme with succinic anhydride was obviously not specific for the amino groups since some hydroxy amino acid esterification occurred. Previously we had shown [30, 32, 33] that succinylation of amino groups in proteins lesulted in some acylation of the aliphatic but not the phenolic hydroxyl groups. It is noteworthy that the lysine residues in lysozyme showed differences in their accessibility to the reagent. The order of accessibility was not entirely that expected from the three-dimensional structure of the protein in the crystalline form [34, 35]. Reactivities decreased in the following order: lys-ll6, Lys-1 (a-NH2), lys-33, lys-96, lys-13 and lys-1 (e-NH2) about equal, lys-97, thr-43. In the crystalline molecular conformation the amino groups have [34-36] the following order of exposure: lys-33 ~ lys-97 lys-1 (e-NH2) ~ lys-96 z lys-ll6 ~ lys-13 ~ lys-1 (a-NH2). The present findings, therefore, do indicate some differences in side-chain accessibility between the conformation of the protein in solution and in the crystalline state. This is not unusual and we have, for example, observed similar differences in the accessibility of the carboxyl groups [37, 14, 15] and of the arginines [13] in lysozyme. Disulfide reducibility revealed some conformational changes in all the derivatives, with the largest alteration being in succinylated derivatives I, | I and III. O R D and CD measurements confirmed the presence of conformational changes in derivative I and may have suggested small changes in derivatives II and III but failed to detect any changes in the other derivatives. Loss of enzymic activity can be attributed

431 to changes in the electrostatic interactions between the modified enzyme and the negatively charged bacterial cell wall, as a result of reversal of charge of the amino groups. We have previously shown in detail [30] that retension of charge of the amino groups is critical for the action of lysozyme against M. Lysodeikticus. In addition, lysine-33 is on the lower end of the substrate-binding cleft of the enzyme, but is not itself involved in binding of the substrate [35]. Lysine-33, however, considerably occludes asparagine-37 [36] which takes active part in binding with the substrate [35, 36]. Therefore, succinylation of lysine-33 should be expected to disturb favorable side-chain orientations within the substrate-binding cleft and will give additional factors for loss of activity of the relevant derivatives. It is perhaps not irrelevant to point out that the X-ray crystallographic studies have primarily studied the activity of lysozyme as a chitinase and its binding with short oligomers of N-acetylglucosamine [35]. It is not unreasonable to expect that some lysine residues on lysozyme may play a larger role [30, 38, 39] in its binding with the long chain, negatively charged polymer of N-acetylglucosamine and N-acetylmuramic acid forming the cell wall of M. Lysodeikticus [40]. Furthermore, results reported here and in our previous work refer to studies in solution and some minor differences between the crystalline and solution structures cannot be excluded (refs. 14, 15, 36, 41, also present work). Since conformational changes were more appreciable in the succinyl derivatives I, II and III, it is best not to base conclusions relating to antigenic structure on these derivatives. It is now well established that conformational changes may influence antigenic reactivity [42-45] and that the immune response to native protein antigens is directed against their native, three-dimensional structure [45, 1, 2]. However, not every conformational change will be expected to exert an influence on the antigenic reactivity [10, 11, 13-15]. The change, or otherwise, in antigenic reactivity will rather depend on the protein and on the nature, location and magnitude of the conformational change. Since conformational changes in the succinyl derivatives IV, V and VI were virtually absent, the present immunochemical findings indicate that lysines-33, -96 and - 116 are part of the antigenic reactive region in lysozyme. Previous studies from our laboratories [3] have shown that the peptides 22-33 (Cys-30--Cys-115) 115-116, 62-68 (Cys-64-Cys-80) 74-96 (Cys-76-Cys-94) and 6--13 (Cys-6-Cys-127) 126-128 account for almost all (90 %) of the immunochemical reaction of the protein. The present findings provide an independent confirmation and further demonstrate that lysines-33, -96 and -116 are essential for binding with antibody. ACKNOWLEDGEMENTS This work was supported by grants (AI 11973 and AI 11974) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service, and by the American Heart Association (Grant No. 71910), and in part by grant (AM 13389) from the National Institutes of Health, U.S. Public Health Service. REFERENCES 1 Atassi, M. Z. (1972) in SpecificReceptors of Antibodies,Antigensand Cells, 3rd Int. Conv. ImmunoI., pp. 118-135, Karger, Basel 2 Atassi, M. Z. (1975) Immunochemistry,12, 423-438

432 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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