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Immunological studies of affinity labelled hen egg-white lysozyme and of the active site region of related lysozymes
BIOCHIMICA ET BIOPHYSICAACTA
243
BBA 36213 IMMUNOLOGICAL STUDIES OF A F F I N I T Y L A B E L L E D HEN E G G - W H I T E LYSOZYME AND OF T H E ACTIVE SITE REGION OF R E L A T E D LYSOZYMES
E L C H A N A N MARON', Y U V A L E S H D A T AND N A T H A N S H A R O N "
Departments of Biophysics and Chemical Immunology, The Weizmann Institute of Science, Rehovoth (Israel) (Received May 3Ist, 1972)
SUMMARY
Hen egg-white lysozyme, irreversibly inhibited by the affinity label 2',3'epoxypropyl /~-glycoside of di(N-acetyl-D-glucosamine) ((GlcNAc)~), reacted with goat anti-hen lysozyme antibodies in the "chemically modified bacteriophage" assay as hen lysozyme and as the enzyme in the presence of a large excess of (GlcNAc)2. Antibodies directed against the active site region of hen lysozyme reacted with the affinity labelled enzyme to a much lesser extent than with hen lysozyme. The reversible complex formed between hen lysozyme and (GlcNAc)2 behaved in the assay with the "active site-directed" antibodies in a manner identical to the affinity labelled enzyme, strongly suggesting that in both cases the (GlcNAc)2 occupies the same site on the enzyme (subsites B and C). The "active site-directed" antibodies were used for the comparison of several other lysozymes and of bovine a-lactalbumin to hen eggwhite lysozyme. Correlations were found between the immunological reactivity of the proteins tested and the differences in their amino acid sequences, in particular with the sequences presumed to be present in the region of their active site.
INTRODUCTION
Hen egg-white lysozyme can be specifically and irreversibly inhibited by the 2',3'-epoxypropyl /5-glycoside of di(N-acetyl-D-glucosamine) ((GlcNAc)2-Ep) 1. The inhibited enzyme ((GlcNAc)2-Pr-lysozyme) has been shown t o contain I mole of (GlcNAc)2 bound covalently via an ester linkage, most probably to the aspartic acid residue in position 52 of the polypeptide chainL X-ray crystallographic studies of (GlcNAc)z-Pr-lysozyme have shown that the (GlcNAc)z moiety of the inhibitor * Present address: D e p a r t m e n t of Microbiology, University of Illinois Medical Center, Chicago, Illinois 60680. *" To whom correspondence should be addressed. Abbreviations: GlcNAc, N-acetyl-D-glucosamine; (GlcNAc),-Ep, 2',3'-epoxypropyl ~glycoside of di(N-acetyl-D-glucosamine); (GlcNAc)2-Pr-lysozyme, hen egg-white lysozyme irreversibly inactivated b y (GlcNAc)2-E p.
Biochim. Biophys. Acta, 278 (1972) 243-249
244
E. MARON et al.
occupies subsites B and C in the active site of the enzyme, and that there are no marked differences in conformation between (GlcNAc)2-Pr-lysozyme and hen lysozyme which had been reversibly inhibited by the fl-phenyl glycoside of (GlcNAc)2 (J. Moult, Y. Eshdat and N. Sharon, unpublished). In addition to inhibiting hen lysozyme specifically and irreversibly, (GlcNAc)2Ep has also been found to inhibit similarly several other bird lysozymes including those from the eggs of the ring-necked pheasant, turkey and duck, as well as human leukemic urine lysozyme (ref. 3 and Y. Eshdat and N. Sharon, unpublished). To the extent that they have been investigated, these enzymes exhibit close similarity in their amino acid sequences 4-7, fluorescence spectra (N. Sharon, E. M. Prager and A. C. Wilson, unpublished; ref. 8) and enzymatic properties9,1°. The latter include not only the ability to digest cell walls of Micrococcus lysodeikticus, but also to catalyse transglycosylation reactions with a variety of saccharide acceptors, when the cell wall tetrasaccharide or chitin oligosaccharides are used as substrates (I. Maoz and N. Sharon, unpublished; ref. II). These observations imply that the general architecture of the active site, and the location and nature of the amino acid side chains which are important for the binding of substrates and inhibitors and for the catalytic processes, are similar in these enzymes. Immunological techniques have recently been employed for the comparison of various bird and human lysozymesg,12-14. Using the highly sensitive "chemically modified bacteriophage" assay15,18, it was possible to detect, in a defined region of the lysozyme molecule, changes as small as the replacement of a single amino acid TM. In the present study, this technique was applied for a comparison of (GlcNAc)2-Prlysozyme, with different lysozymes and with bovine a-lactalbumin, by measuring their ability to inhibit the immunological reaction between hen lysozyme and antibodies directed against the active site region of hen lysozymO 7 ("active site-directed" antibodies). The results are discussed in terms of sequence differences between the various lysozymes, in particular in their presumed active site regions. MATERIALS AND METHODS
Hen egg-white lysozyme was purchased from Worthington; bovine a-lactalbumin was obtained from Sigma. Human leukemic urine lysozyme was a gift of Dr R. E. Canfield; ring-necked pheasant lysozyme and turkey lysozyme were generously provided by Dr A. C. Wilson and Dr Ellen M. Prager; duck II lysozyme was kindly given to us by Dr P. Joll~s. Oligosaccharides of N-acetyl-D-glucosamine ((GlcNAc)n) were obtained according to the general procedure described by Barker et al. TM. (GlcNAc)2-Ep was prepared according to the method of Thomas 19. (GlcNAc)2-Pr-lysozyme was obtained by the incubation of IOO mg of hen lysozyme and 50 mg of (GlcNAc)~-Ep in 5 ° ml of water at 37 °C for 24 h. The incubation mixture was dialysed exhaustively against water and the non-dialysable material was further purified by gel filtration on a Sephadex G-25 (fine) column (2 cm × 15° cm) in o.I M. ammonium acetate. The fractions containing the protein were combined and dialysed against water. After lyophilization, the isolated protein was found to be homogeneous by disc electrophoresis at pH 4-5- Its enzymatic activity, when assayed with cells of M . lysodeikticus 24 was found to be less than 2% of that of an equal weight of hen lysozyme. Analysis of Biochlm. Biophys. Acta, 278 (1972) 243-249
245
AFFINITY LABELLED LYSOZYME
the acid hydrolysate (6 M HC1, IiO °C, 22 h) of (GlcNAc)2-Pr-lysozyme on an amino acid analyzer revealed, in addition to the expected amino acids the presence of glucosamine in a ratio of 1. 9 moles per mole of enzyme. Antibodies against hen lysozyme were obtained in goats and were purified by using a hen lysozyme immunoadsorbent 21. "Active site-directed" antibodies to hen lysozyme were prepared according to the procedure described by Imanishi et alY, by dissociating the complex formed between hen lysozyme and the anti-hen lysozyme antibodies using a mixture of oligosaccharides of GlcNAc. The immunological reactivity of the various lysozymes, with the two types of antibody, was assayed by measuring their ability to inhibit the inactivation of a hen lysozyme-bacteriophage T, conjugate by free antibodies 2~. Antibodies to hen lysozyme (1. 4. lO -5 mg per sample), or the "active site-directed" antibodies (8. 4. lO -4 mg per sample), were kept for 24 h at 4 °C, with different concentrations of the various proteins examined. The hen lysozyme-bacteriophage T 4 conjugate preparation was then added, and allowed to react with the antibody-protein mixture for 2 h prior to plating on an agar layer and scoring the number of survivors. The extent of inhibition was calculated from the extent of inactivation in the presence and in the absence of the inhibitory protein. The antibody concentration was determined spectrophotometrically at 280 nm X(~I% _-- 14.o). Solutions of the different enzymes were --I cm prepared by dissolving a known weight of the dry material. RESULTS
Two different populations of antibodies directed against hen lysozyme were used in this study. The first contained a mixture of antibodies formed against all the antigenic determinants present on the enzyme molecule; the second, directed mainly against the active site region 17, was composed of antibodies whose reaction with lysozyme was inhibited by oligosaccharides of GlcNAc. Both antibody preparations interact with hen lysozyme specifically and at very low concentrations, as manifested by the inactivation of the hen lysozyme-bacteriophage conjugate. The results
A•
I
I
I
i
B
I
I
100
I
1
0~0
I
/////&
80 60
/ /
"3
~40
.C C
~0
I
10-7
1o- 5
I
10-7 10-5 1o 3 Lysozyrne added (mg)
"ll/'i' I 10-3
L
10-1
P
Fig. I. Inhibition of the inactivation of hen lysozyme-bacteriophage T 4 conjugate by goat antihen lysozyme antibodies (A) and by anti-hen lysozyme "active site-directed" antibodies (B) in the presence of hen lysozyme, (GlcNAc)2-Pr-lysozyme and lysozyme-(GlcNAc)2 reversible complex. The following symbols are used: © - - O hen egg-white lysozyme; 0 - - - 0 , (GlcNAc)2-Prlysozyme; ~ - - - A , lysozyme-(GlcNAc), (I :4 ° molar ratio). Biochim. Biophys. Acta, 278 (1972) 243-249
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E. MARON et al.
of the inhibition of the inactivation, caused by the addition of hen lysozyme, or of its affinity labelled derivative (GlcNAc)~-Pr-lysozyme, are illustrated in Fig. i. Results obtained when hen lysozyme and a large excess of (GlcNAc)2 (molar ratio of enzyme to saccharide i :4o) were used, are also given in Fig. I. It can be seen that when antibodies to the whole enzyme molecule were used, the inhibitory capacity of the native and the chemically modified proteins was indistinguishable and the presence of excess of (GlcNAc)2 had no effect on the interaction of the enzyme with the antibodies (Fig. IA). However, when the "active site-directed" antibodies were used for the assay, both the reversible complex of (GlcNAc)2 and hen lysozyme, and (GlcNAc)2-Pr-lysozyme, had 15o times weaker inhibitory capacity than hen lysozyme (Fig. IB). The same system served for the examination of the degree of immunological similarity between the active site region of hen lysozyme and other lysozymes. With the "active site-directed" antibodies, it was found that duck lysozyme was 25-fold less active as inhibitor than hen lysozyme, and pheasant lysozyme was 70-fold less active; turkey lysozyme had much weaker inhibitory capacity, namely 46o-fold less then hen lysozyme. When a large excess of (GlcNAc)2 was added to duck lysozyme, its inhibitory activity was reduced by a factor of only 2 (Fig. 2B). -A
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;
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I
l
I
o%
~4C _o
10-7
10-5
10-3
10-7
10"5
10:'3
10-1
Lysozyme added (rag) Fig. 2. Inhibition of the inactivation of hen lysozyme-bacteriophage T 4 conjugate b y goat antihen lysozyme antibodies (A) and by anti-hen lysozyme "active site-directed" antibodies (B), caused b y different lysozymes and bovine a-lactalbumin. The following symbols are used: (2)--@, hen lysozyme; V - - V , turkey lysozyme; ~ - - / ~ , ring-necked p h e a s a n t lysozyme; [ ] - - [ B , duck 11 lysozyme; i - - i , duck II lysozyme-(GlcNAc)2 (I :4 ° molar ratio); × - - × , h u m a n lysozyme; 0--~), bovine a-lactalbumin.
A completely different pattern of reactivity was observed when the different proteins were assayed with antibodies against the whole molecule of hen lysozyme. Compared to hen lysozyme, turkey lysozyme was only 8-fold less efficient as an inhibitor, whereas pheasant lysozyme was 5o times less, and duck lysozyme was 32o-fold less efficient (Fig. 2A). Bovine a-lactalbumin and the lysozyme of human origin did not react with the antibodies in any of the systems tested by us (Fig. 2A, B). DISCUSSION
Antibodies directed against the active site region of hen lysozyme compete with oligosaccharides of GlcNAc for the binding to the enzyme 17. These "active siteBiochim. Biophys. Acta, 278 (1972) 243-249
AFFINITY LABELLED LYSOZYME
247
directed" antibodies served, in the present study, for the detection of changes which occur in that particular region of hen lysozyme and of related proteins, either by chemical modification or as a result of genetic changes. As shown in Fig. IA, hen lysozyme, (GlcNAc)2-Pr-lysozyme, and hen lysozyme in the presence of excess of (GlcNAc)z, react very similarly when assayed with antibodies to the whole enzyme. Thus, under our experimental conditions, changes in the active site region of hen lysozyme did not affect to any significant extent the interaction of the enzyme with anti-hen lysozyme antibodies. This is in contrast with the findings reported by von Fellenberg and Levine 22, who, using the micro-complement fixation method, found that (GlcNAc)3 inhibited markedly the hen lysozyme-anti-hen lysozyme reaction. In order to follow changes in the active site of hen lysozyme and of related proteins, it was necessary to use "active site-directed" antibodies 17. Introduction of (GlcNAc)2 by reversible association, to subsites B and C in the active site of hen lysozyme, led to a marked decrease in the interaction of the protein with these antibodies (Fig. IB). An identical pattern is also shown for the affinity labelled enzyme E(GlcNAc)i-Pr-lysozyme1. This finding is in agreement with the suggestion, 1 that the (GlcNAc)2 moiety of the affinity label occupies subsites B and C in the active site of hen lysozyme, and is in line with our recent X-ray crystallographic studies of (GlcNAc)2-Pr-lysozyme (J. Moult, Y. Eshdat and N. Sharon, unpublished). The different proteins tested show great similarity in their enzymatic activities and amino acid sequencesS-7,",1°, particularly in the amino acids which correspond to the residues which make contact with the substrate in the hen lysozyme-substrate complex 2~ (Table I). It could therefore be expected that the immunological reactivity of the "active site-directed" antibodies, with the different proteins, would be quite similar. Tile results obtained were not in full agreement with such an expectation. The enzymes tested differed in the extent of interaction with the "active site-directed" antibodies, and in the experiments with turkey lysozyme, this interaction was even weaker than with the anti-hen lysozyme antibodies. Also, the addition of (GlcNAc), to the duck lysozyme decreased its interaction with these antibodies only by a factor of 2. Therefore, it may be assumed that the "active site-directed" antibodies are specific not only for contact amino acids, but for an "active site region", which includes the amino acids lining the lysozyme cleft, as well as neighbouring surface amino acids. This was already noted by Imanishi et al. 1~ who concluded that the "active site-directed" antibodies are indeed also specific to other regions since they are "precipitating antibodies". In addition, the conformations of the various enzymes may differ, and such differences may also affect their immunological cross reactions. However, a partial explanation for our results can be derived by considering only the "contact" amino acid residues, i.e. those residues which in the different lysozymes and in a-lactalbumin occupy positions corresponding to the amino acid residues which are within 4 A from substrate atoms in the hen lysozyme-substrate complex za (Table I). The interaction of turkey lysozyme with anti-hen lysozyme antibodies is about 6o-fold stronger than its interaction with the "anti-hen lysozyme active site-directed" antibodies. Turkey lysozyme has the same number of amino acid residues as has hen lysozyme but there are seven differences in the amino acid sequences of these two Biochim. Biophys. Acta, 278 (1972) 243-249
248
~. ~ARON et al.
TABLE I "CONTACT AMINO A C I D S " * IN D I F F E R E N T L Y S O Z Y M E S AND IN ~ - L A C T A L B U M I N
Protein
Total residues
Residue No.*" 35
44
52
57
58
59
62
63
~oI ~o3 zo7 zo8 xo9 Tz4
Number of sequence differences compared to hen lysozyme Contact amino acids
Complete molecule
Hen
lysozyme23 Turkey lysozyme e Duck II lysozyme5 Human lysozyme 7 a-Lactalb u m i n 25
129
Glu Asn Asp Gin Ile
Asn Trp Trp Asp Asp Ala Trp Val Arg
--
129
Glu Asn Asp Gln IIe Asn Trp Trp Gly Asp Ala Trp Val Arg
1
7
129
Glu Asn Asp Glu Ile
Asn Trp Trp Asp Asp Ala Trp Val Arg
I
22
129
Glu Ash Asp Gln Ile
Asn Tyr Trp Asp Gin Ala Trp Val Arg
2
53
I24
His Val Asp Gln Ile
Asn Ile
7
80
Trp Asp Val Tyr Trp Leu Leu
* H a v i n g side chains which in the hen l y s o z y m e - s u b s t r a t e complex h a v e a t o m s within 4 A of s u b s t r a t e a t o m s 23. ** Aligned according to the sequence of hen lysozyme. Replacements of amino acids with respect to the h e n lysozyme sequences are in italics.
proteins e. Although the proportions of the changes in the active site region and in the whole molecule are about the same (Table I), the single change in the active site is a very marked one : it involves the replacement of Asp IOi, which in hen lysozyme is highly exposed and participates in substrate binding23, ~4, by a glycyl residue in turkey lysozyme. This change is most probably the main reason for the weakening of the interaction of turkey lysozyme with the "active site-directed" antibodies, compared to its interaction with the anti-hen lysozyme antibodies. On the other hand, the interaction of duck lysozyme with the "active sitedirected" antibodies is I3-fold stronger than its interaction with the anti-hen lysozyme antibodies. The total number of sequence differences between duck lysozyme and hen lysozyme is 22 (out of 129 amino acids), while the number of changes in the contact amino acids region is very small--one out of 14 amino acids. This single change involves the replacement of Gln 57, a deeply buried residue in hen lysozyme, by a glutamic acid residue. Therefore, it is not surprising that the interaction of duck lysozyme and the anti-hen lysozyme "active site-directed" antibodies is stronger than its interaction with the anti-hen lysozyme antibodies. Pheasant lysozyme interacts to about the same extent with both the anti-hen lysozyme antibodies and the "active site-directed" antibodies. In both cases, the reaction is much weaker than that of the same antibodies with hen lysozyme. Since the primary structure of pheasant lysozyme is not yet known, we can only assume that the seven differences found between the amino acids of pheasant lysozyme and hen lysozyme 13 are equally distributed in the active site region and in the other parts of the molecule, thus affecting to approximately the same extent the interaction of the two populations of antibodies with the pheasant lysozyme molecule. The amino acid sequence of human lysozyme differs markedly from that of hen lysozyme (53 residues out of 129). Two of these differences occur in positions corresBiochim. Biophys. Acta, 278 (1972) 243-249
AFFINITY LABELLED LYSOZYME
249
ponding to those of the contact amino acids in hen lysozyme (Trp 62 --~ Tyr 63 and Asp lO3 -+ Gln lO3) and may by themselves be sufficient to cause the lack of reactivity of this enzyme with the "active site-directed" antibodies. However, it is very likely that differences in the amino acids near the active site also contribute to this lack of reactivity. Although a-lactalbumin is believed to have a structure similar to that of hen lysozyme2a,25, it does not cross-react with antibodies to hen lysozyme ~6 or with the anti-hen lysozyme "active site-directed" antibodies. If indeed a-lactalbumin has a surface cleft similar to part of the hen lysozyme cleft ~a, it seems that the two clefts are immunologically distinct. ACKNOWLEDGEMENTS
The authors wish to thank the following people for the gifts of the different lysozymes. Dr R. E. Canfield of the Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, for the human leukemic urine lysozyme ; Dr A. C. Wilson and Dr Ellen M. Prager of the Department of Biochemistry, University of California, Berkeley, for the ring-necked pheasant and turkey lysozymes; and Dr P. Joll~s of the Laboratory of Biochemistry, Faculty of Sciences, University of Paris, for the duck II lysozyme. REFERENCES
i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
E. W. Thomas, J. F. M c K e l v y a n d N. Sharon, Nature, 222 (1969) 485 • J. F. McKelvy, Y. E s h d a t a n d N. Sharon, Israel J. Chem., 8 (197 o) i7o p. J. F. McKelvy, Y. E s h d a t a n d N. Sharon, Fed. Proc., 29 (197 o) 532. M. K a n e d a , I. Kato, N. Tominaga, K. Titani a n d K. Narita, J. Biochem. Tokyo, 66 (1969) 747. J. H e r m a n n a n d J. Joll~s, Biochim. Biophys. Acta, 200 (197 o) 178. J. N. L a R u e and J. C. Speck, Jr, J. Biol. Chem., 245 (197 ° ) 1985. R. E. Canfield, S. K a m m e r m a n , J. H. Sobel a n d F. J. Morgan, Nature New Biol., 232 (1971) 16. J. Saint-Blancard, A. Capbern, D. Ducass6 a n d P. Joll~s, C.R. Acad. Sci. Paris, 269 (1969) 858. E. M. P r a g e r and A. C. Wilson, J. Biol. Chem., 246 (1971) 5978. J. P. Locquet, J. S a i n t - B l a n c a r d a n d P. Joll~s, Biochim. Biophys. Acta, 167 (1968) 15o. N. Sharon, J. Jollbs and P. Joll~s, Bull. Soc. Chim. Biol., 48 (1966) 731. E. Maron, R. Arnon, M. Sela, J. P. P e r i n a n d P. Joll~s, Biochim. Biophys. Acta, 214 (197 o) 222. N. Arnheim, E. M. P r a g e r a n d A. C. Wilson, J. Biol. Chem., 244 (1969) 2085. E. M. P r a g e r a n d A. C. Wilson, J. Biol. Chem., 246 (1971) 7OLO. J. Haimovich, E. Hurwitz, N. Novik a n d M. Sela, Biochim. Biophys. Acta, 207 (197 o) 115. J. Haimovich, E. Hurwitz, N. Novik a n d M. Sela, Biochim. Biophys. Acta, 207 (197 o) 125. M. h n a n i s h i , N. Miyagawa, H. Fujio a n d T. A m a n o , Biken J., 12 (1969) 85. S. A. Barker, A. B. Foster, M. Stacey and J. M. W e b b e r , J. Chem. Soc., (1958) 2218. E. W. Thomas, Carbohydr. Res., 13 (197 o) 225. D. Shugar, Biochim. Biophys. Acta, 8 (1952) 302. E. Maron, C. Shiozawa, R. A r n o n a n d M. Sela, Biochemistry, IO (1971) 763. R. von Fellenberg a n d L. Levine, Immunochemistry, 4 (1967) 363 . W. J. Browne, A. C. T. North, D. C. Phillips, K. Brew, T. C. V a n a m a n and R. L. Hill, J. Mol. Biol., 42 (1969) 65. G. P. Hess a n d J. A. Rupley, Annu. Rev. Biochem., 4 ° (1971 ) lOl 3. T. C. V a n a m a n , K. Brew a n d R. L. Hill, J. Biol. Chem., 245 (197 o) 4583 . R. A r n o n a n d E. Maron, J. Mol. Biol., 51 (197 o) 7o3 .
Biochim. Biophys. A cta, 278 (i972) 243-249
243
BBA 36213 IMMUNOLOGICAL STUDIES OF A F F I N I T Y L A B E L L E D HEN E G G - W H I T E LYSOZYME AND OF T H E ACTIVE SITE REGION OF R E L A T E D LYSOZYMES
E L C H A N A N MARON', Y U V A L E S H D A T AND N A T H A N S H A R O N "
Departments of Biophysics and Chemical Immunology, The Weizmann Institute of Science, Rehovoth (Israel) (Received May 3Ist, 1972)
SUMMARY
Hen egg-white lysozyme, irreversibly inhibited by the affinity label 2',3'epoxypropyl /~-glycoside of di(N-acetyl-D-glucosamine) ((GlcNAc)~), reacted with goat anti-hen lysozyme antibodies in the "chemically modified bacteriophage" assay as hen lysozyme and as the enzyme in the presence of a large excess of (GlcNAc)2. Antibodies directed against the active site region of hen lysozyme reacted with the affinity labelled enzyme to a much lesser extent than with hen lysozyme. The reversible complex formed between hen lysozyme and (GlcNAc)2 behaved in the assay with the "active site-directed" antibodies in a manner identical to the affinity labelled enzyme, strongly suggesting that in both cases the (GlcNAc)2 occupies the same site on the enzyme (subsites B and C). The "active site-directed" antibodies were used for the comparison of several other lysozymes and of bovine a-lactalbumin to hen eggwhite lysozyme. Correlations were found between the immunological reactivity of the proteins tested and the differences in their amino acid sequences, in particular with the sequences presumed to be present in the region of their active site.
INTRODUCTION
Hen egg-white lysozyme can be specifically and irreversibly inhibited by the 2',3'-epoxypropyl /5-glycoside of di(N-acetyl-D-glucosamine) ((GlcNAc)2-Ep) 1. The inhibited enzyme ((GlcNAc)2-Pr-lysozyme) has been shown t o contain I mole of (GlcNAc)2 bound covalently via an ester linkage, most probably to the aspartic acid residue in position 52 of the polypeptide chainL X-ray crystallographic studies of (GlcNAc)z-Pr-lysozyme have shown that the (GlcNAc)z moiety of the inhibitor * Present address: D e p a r t m e n t of Microbiology, University of Illinois Medical Center, Chicago, Illinois 60680. *" To whom correspondence should be addressed. Abbreviations: GlcNAc, N-acetyl-D-glucosamine; (GlcNAc),-Ep, 2',3'-epoxypropyl ~glycoside of di(N-acetyl-D-glucosamine); (GlcNAc)2-Pr-lysozyme, hen egg-white lysozyme irreversibly inactivated b y (GlcNAc)2-E p.
Biochim. Biophys. Acta, 278 (1972) 243-249
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E. MARON et al.
occupies subsites B and C in the active site of the enzyme, and that there are no marked differences in conformation between (GlcNAc)2-Pr-lysozyme and hen lysozyme which had been reversibly inhibited by the fl-phenyl glycoside of (GlcNAc)2 (J. Moult, Y. Eshdat and N. Sharon, unpublished). In addition to inhibiting hen lysozyme specifically and irreversibly, (GlcNAc)2Ep has also been found to inhibit similarly several other bird lysozymes including those from the eggs of the ring-necked pheasant, turkey and duck, as well as human leukemic urine lysozyme (ref. 3 and Y. Eshdat and N. Sharon, unpublished). To the extent that they have been investigated, these enzymes exhibit close similarity in their amino acid sequences 4-7, fluorescence spectra (N. Sharon, E. M. Prager and A. C. Wilson, unpublished; ref. 8) and enzymatic properties9,1°. The latter include not only the ability to digest cell walls of Micrococcus lysodeikticus, but also to catalyse transglycosylation reactions with a variety of saccharide acceptors, when the cell wall tetrasaccharide or chitin oligosaccharides are used as substrates (I. Maoz and N. Sharon, unpublished; ref. II). These observations imply that the general architecture of the active site, and the location and nature of the amino acid side chains which are important for the binding of substrates and inhibitors and for the catalytic processes, are similar in these enzymes. Immunological techniques have recently been employed for the comparison of various bird and human lysozymesg,12-14. Using the highly sensitive "chemically modified bacteriophage" assay15,18, it was possible to detect, in a defined region of the lysozyme molecule, changes as small as the replacement of a single amino acid TM. In the present study, this technique was applied for a comparison of (GlcNAc)2-Prlysozyme, with different lysozymes and with bovine a-lactalbumin, by measuring their ability to inhibit the immunological reaction between hen lysozyme and antibodies directed against the active site region of hen lysozymO 7 ("active site-directed" antibodies). The results are discussed in terms of sequence differences between the various lysozymes, in particular in their presumed active site regions. MATERIALS AND METHODS
Hen egg-white lysozyme was purchased from Worthington; bovine a-lactalbumin was obtained from Sigma. Human leukemic urine lysozyme was a gift of Dr R. E. Canfield; ring-necked pheasant lysozyme and turkey lysozyme were generously provided by Dr A. C. Wilson and Dr Ellen M. Prager; duck II lysozyme was kindly given to us by Dr P. Joll~s. Oligosaccharides of N-acetyl-D-glucosamine ((GlcNAc)n) were obtained according to the general procedure described by Barker et al. TM. (GlcNAc)2-Ep was prepared according to the method of Thomas 19. (GlcNAc)2-Pr-lysozyme was obtained by the incubation of IOO mg of hen lysozyme and 50 mg of (GlcNAc)~-Ep in 5 ° ml of water at 37 °C for 24 h. The incubation mixture was dialysed exhaustively against water and the non-dialysable material was further purified by gel filtration on a Sephadex G-25 (fine) column (2 cm × 15° cm) in o.I M. ammonium acetate. The fractions containing the protein were combined and dialysed against water. After lyophilization, the isolated protein was found to be homogeneous by disc electrophoresis at pH 4-5- Its enzymatic activity, when assayed with cells of M . lysodeikticus 24 was found to be less than 2% of that of an equal weight of hen lysozyme. Analysis of Biochlm. Biophys. Acta, 278 (1972) 243-249
245
AFFINITY LABELLED LYSOZYME
the acid hydrolysate (6 M HC1, IiO °C, 22 h) of (GlcNAc)2-Pr-lysozyme on an amino acid analyzer revealed, in addition to the expected amino acids the presence of glucosamine in a ratio of 1. 9 moles per mole of enzyme. Antibodies against hen lysozyme were obtained in goats and were purified by using a hen lysozyme immunoadsorbent 21. "Active site-directed" antibodies to hen lysozyme were prepared according to the procedure described by Imanishi et alY, by dissociating the complex formed between hen lysozyme and the anti-hen lysozyme antibodies using a mixture of oligosaccharides of GlcNAc. The immunological reactivity of the various lysozymes, with the two types of antibody, was assayed by measuring their ability to inhibit the inactivation of a hen lysozyme-bacteriophage T, conjugate by free antibodies 2~. Antibodies to hen lysozyme (1. 4. lO -5 mg per sample), or the "active site-directed" antibodies (8. 4. lO -4 mg per sample), were kept for 24 h at 4 °C, with different concentrations of the various proteins examined. The hen lysozyme-bacteriophage T 4 conjugate preparation was then added, and allowed to react with the antibody-protein mixture for 2 h prior to plating on an agar layer and scoring the number of survivors. The extent of inhibition was calculated from the extent of inactivation in the presence and in the absence of the inhibitory protein. The antibody concentration was determined spectrophotometrically at 280 nm X(~I% _-- 14.o). Solutions of the different enzymes were --I cm prepared by dissolving a known weight of the dry material. RESULTS
Two different populations of antibodies directed against hen lysozyme were used in this study. The first contained a mixture of antibodies formed against all the antigenic determinants present on the enzyme molecule; the second, directed mainly against the active site region 17, was composed of antibodies whose reaction with lysozyme was inhibited by oligosaccharides of GlcNAc. Both antibody preparations interact with hen lysozyme specifically and at very low concentrations, as manifested by the inactivation of the hen lysozyme-bacteriophage conjugate. The results
A•
I
I
I
i
B
I
I
100
I
1
0~0
I
/////&
80 60
/ /
"3
~40
.C C
~0
I
10-7
1o- 5
I
10-7 10-5 1o 3 Lysozyrne added (mg)
"ll/'i' I 10-3
L
10-1
P
Fig. I. Inhibition of the inactivation of hen lysozyme-bacteriophage T 4 conjugate by goat antihen lysozyme antibodies (A) and by anti-hen lysozyme "active site-directed" antibodies (B) in the presence of hen lysozyme, (GlcNAc)2-Pr-lysozyme and lysozyme-(GlcNAc)2 reversible complex. The following symbols are used: © - - O hen egg-white lysozyme; 0 - - - 0 , (GlcNAc)2-Prlysozyme; ~ - - - A , lysozyme-(GlcNAc), (I :4 ° molar ratio). Biochim. Biophys. Acta, 278 (1972) 243-249
246
E. MARON et al.
of the inhibition of the inactivation, caused by the addition of hen lysozyme, or of its affinity labelled derivative (GlcNAc)~-Pr-lysozyme, are illustrated in Fig. i. Results obtained when hen lysozyme and a large excess of (GlcNAc)2 (molar ratio of enzyme to saccharide i :4o) were used, are also given in Fig. I. It can be seen that when antibodies to the whole enzyme molecule were used, the inhibitory capacity of the native and the chemically modified proteins was indistinguishable and the presence of excess of (GlcNAc)2 had no effect on the interaction of the enzyme with the antibodies (Fig. IA). However, when the "active site-directed" antibodies were used for the assay, both the reversible complex of (GlcNAc)2 and hen lysozyme, and (GlcNAc)2-Pr-lysozyme, had 15o times weaker inhibitory capacity than hen lysozyme (Fig. IB). The same system served for the examination of the degree of immunological similarity between the active site region of hen lysozyme and other lysozymes. With the "active site-directed" antibodies, it was found that duck lysozyme was 25-fold less active as inhibitor than hen lysozyme, and pheasant lysozyme was 70-fold less active; turkey lysozyme had much weaker inhibitory capacity, namely 46o-fold less then hen lysozyme. When a large excess of (GlcNAc)2 was added to duck lysozyme, its inhibitory activity was reduced by a factor of only 2 (Fig. 2B). -A
I--N
i -- I
100
;
I _B
8O
o~v// l
I
l
I
o%
~4C _o
10-7
10-5
10-3
10-7
10"5
10:'3
10-1
Lysozyme added (rag) Fig. 2. Inhibition of the inactivation of hen lysozyme-bacteriophage T 4 conjugate b y goat antihen lysozyme antibodies (A) and by anti-hen lysozyme "active site-directed" antibodies (B), caused b y different lysozymes and bovine a-lactalbumin. The following symbols are used: (2)--@, hen lysozyme; V - - V , turkey lysozyme; ~ - - / ~ , ring-necked p h e a s a n t lysozyme; [ ] - - [ B , duck 11 lysozyme; i - - i , duck II lysozyme-(GlcNAc)2 (I :4 ° molar ratio); × - - × , h u m a n lysozyme; 0--~), bovine a-lactalbumin.
A completely different pattern of reactivity was observed when the different proteins were assayed with antibodies against the whole molecule of hen lysozyme. Compared to hen lysozyme, turkey lysozyme was only 8-fold less efficient as an inhibitor, whereas pheasant lysozyme was 5o times less, and duck lysozyme was 32o-fold less efficient (Fig. 2A). Bovine a-lactalbumin and the lysozyme of human origin did not react with the antibodies in any of the systems tested by us (Fig. 2A, B). DISCUSSION
Antibodies directed against the active site region of hen lysozyme compete with oligosaccharides of GlcNAc for the binding to the enzyme 17. These "active siteBiochim. Biophys. Acta, 278 (1972) 243-249
AFFINITY LABELLED LYSOZYME
247
directed" antibodies served, in the present study, for the detection of changes which occur in that particular region of hen lysozyme and of related proteins, either by chemical modification or as a result of genetic changes. As shown in Fig. IA, hen lysozyme, (GlcNAc)2-Pr-lysozyme, and hen lysozyme in the presence of excess of (GlcNAc)z, react very similarly when assayed with antibodies to the whole enzyme. Thus, under our experimental conditions, changes in the active site region of hen lysozyme did not affect to any significant extent the interaction of the enzyme with anti-hen lysozyme antibodies. This is in contrast with the findings reported by von Fellenberg and Levine 22, who, using the micro-complement fixation method, found that (GlcNAc)3 inhibited markedly the hen lysozyme-anti-hen lysozyme reaction. In order to follow changes in the active site of hen lysozyme and of related proteins, it was necessary to use "active site-directed" antibodies 17. Introduction of (GlcNAc)2 by reversible association, to subsites B and C in the active site of hen lysozyme, led to a marked decrease in the interaction of the protein with these antibodies (Fig. IB). An identical pattern is also shown for the affinity labelled enzyme E(GlcNAc)i-Pr-lysozyme1. This finding is in agreement with the suggestion, 1 that the (GlcNAc)2 moiety of the affinity label occupies subsites B and C in the active site of hen lysozyme, and is in line with our recent X-ray crystallographic studies of (GlcNAc)2-Pr-lysozyme (J. Moult, Y. Eshdat and N. Sharon, unpublished). The different proteins tested show great similarity in their enzymatic activities and amino acid sequencesS-7,",1°, particularly in the amino acids which correspond to the residues which make contact with the substrate in the hen lysozyme-substrate complex 2~ (Table I). It could therefore be expected that the immunological reactivity of the "active site-directed" antibodies, with the different proteins, would be quite similar. Tile results obtained were not in full agreement with such an expectation. The enzymes tested differed in the extent of interaction with the "active site-directed" antibodies, and in the experiments with turkey lysozyme, this interaction was even weaker than with the anti-hen lysozyme antibodies. Also, the addition of (GlcNAc), to the duck lysozyme decreased its interaction with these antibodies only by a factor of 2. Therefore, it may be assumed that the "active site-directed" antibodies are specific not only for contact amino acids, but for an "active site region", which includes the amino acids lining the lysozyme cleft, as well as neighbouring surface amino acids. This was already noted by Imanishi et al. 1~ who concluded that the "active site-directed" antibodies are indeed also specific to other regions since they are "precipitating antibodies". In addition, the conformations of the various enzymes may differ, and such differences may also affect their immunological cross reactions. However, a partial explanation for our results can be derived by considering only the "contact" amino acid residues, i.e. those residues which in the different lysozymes and in a-lactalbumin occupy positions corresponding to the amino acid residues which are within 4 A from substrate atoms in the hen lysozyme-substrate complex za (Table I). The interaction of turkey lysozyme with anti-hen lysozyme antibodies is about 6o-fold stronger than its interaction with the "anti-hen lysozyme active site-directed" antibodies. Turkey lysozyme has the same number of amino acid residues as has hen lysozyme but there are seven differences in the amino acid sequences of these two Biochim. Biophys. Acta, 278 (1972) 243-249
248
~. ~ARON et al.
TABLE I "CONTACT AMINO A C I D S " * IN D I F F E R E N T L Y S O Z Y M E S AND IN ~ - L A C T A L B U M I N
Protein
Total residues
Residue No.*" 35
44
52
57
58
59
62
63
~oI ~o3 zo7 zo8 xo9 Tz4
Number of sequence differences compared to hen lysozyme Contact amino acids
Complete molecule
Hen
lysozyme23 Turkey lysozyme e Duck II lysozyme5 Human lysozyme 7 a-Lactalb u m i n 25
129
Glu Asn Asp Gin Ile
Asn Trp Trp Asp Asp Ala Trp Val Arg
--
129
Glu Asn Asp Gln IIe Asn Trp Trp Gly Asp Ala Trp Val Arg
1
7
129
Glu Asn Asp Glu Ile
Asn Trp Trp Asp Asp Ala Trp Val Arg
I
22
129
Glu Ash Asp Gln Ile
Asn Tyr Trp Asp Gin Ala Trp Val Arg
2
53
I24
His Val Asp Gln Ile
Asn Ile
7
80
Trp Asp Val Tyr Trp Leu Leu
* H a v i n g side chains which in the hen l y s o z y m e - s u b s t r a t e complex h a v e a t o m s within 4 A of s u b s t r a t e a t o m s 23. ** Aligned according to the sequence of hen lysozyme. Replacements of amino acids with respect to the h e n lysozyme sequences are in italics.
proteins e. Although the proportions of the changes in the active site region and in the whole molecule are about the same (Table I), the single change in the active site is a very marked one : it involves the replacement of Asp IOi, which in hen lysozyme is highly exposed and participates in substrate binding23, ~4, by a glycyl residue in turkey lysozyme. This change is most probably the main reason for the weakening of the interaction of turkey lysozyme with the "active site-directed" antibodies, compared to its interaction with the anti-hen lysozyme antibodies. On the other hand, the interaction of duck lysozyme with the "active sitedirected" antibodies is I3-fold stronger than its interaction with the anti-hen lysozyme antibodies. The total number of sequence differences between duck lysozyme and hen lysozyme is 22 (out of 129 amino acids), while the number of changes in the contact amino acids region is very small--one out of 14 amino acids. This single change involves the replacement of Gln 57, a deeply buried residue in hen lysozyme, by a glutamic acid residue. Therefore, it is not surprising that the interaction of duck lysozyme and the anti-hen lysozyme "active site-directed" antibodies is stronger than its interaction with the anti-hen lysozyme antibodies. Pheasant lysozyme interacts to about the same extent with both the anti-hen lysozyme antibodies and the "active site-directed" antibodies. In both cases, the reaction is much weaker than that of the same antibodies with hen lysozyme. Since the primary structure of pheasant lysozyme is not yet known, we can only assume that the seven differences found between the amino acids of pheasant lysozyme and hen lysozyme 13 are equally distributed in the active site region and in the other parts of the molecule, thus affecting to approximately the same extent the interaction of the two populations of antibodies with the pheasant lysozyme molecule. The amino acid sequence of human lysozyme differs markedly from that of hen lysozyme (53 residues out of 129). Two of these differences occur in positions corresBiochim. Biophys. Acta, 278 (1972) 243-249
AFFINITY LABELLED LYSOZYME
249
ponding to those of the contact amino acids in hen lysozyme (Trp 62 --~ Tyr 63 and Asp lO3 -+ Gln lO3) and may by themselves be sufficient to cause the lack of reactivity of this enzyme with the "active site-directed" antibodies. However, it is very likely that differences in the amino acids near the active site also contribute to this lack of reactivity. Although a-lactalbumin is believed to have a structure similar to that of hen lysozyme2a,25, it does not cross-react with antibodies to hen lysozyme ~6 or with the anti-hen lysozyme "active site-directed" antibodies. If indeed a-lactalbumin has a surface cleft similar to part of the hen lysozyme cleft ~a, it seems that the two clefts are immunologically distinct. ACKNOWLEDGEMENTS
The authors wish to thank the following people for the gifts of the different lysozymes. Dr R. E. Canfield of the Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, for the human leukemic urine lysozyme ; Dr A. C. Wilson and Dr Ellen M. Prager of the Department of Biochemistry, University of California, Berkeley, for the ring-necked pheasant and turkey lysozymes; and Dr P. Joll~s of the Laboratory of Biochemistry, Faculty of Sciences, University of Paris, for the duck II lysozyme. REFERENCES
i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
E. W. Thomas, J. F. M c K e l v y a n d N. Sharon, Nature, 222 (1969) 485 • J. F. McKelvy, Y. E s h d a t a n d N. Sharon, Israel J. Chem., 8 (197 o) i7o p. J. F. McKelvy, Y. E s h d a t a n d N. Sharon, Fed. Proc., 29 (197 o) 532. M. K a n e d a , I. Kato, N. Tominaga, K. Titani a n d K. Narita, J. Biochem. Tokyo, 66 (1969) 747. J. H e r m a n n a n d J. Joll~s, Biochim. Biophys. Acta, 200 (197 o) 178. J. N. L a R u e and J. C. Speck, Jr, J. Biol. Chem., 245 (197 ° ) 1985. R. E. Canfield, S. K a m m e r m a n , J. H. Sobel a n d F. J. Morgan, Nature New Biol., 232 (1971) 16. J. Saint-Blancard, A. Capbern, D. Ducass6 a n d P. Joll~s, C.R. Acad. Sci. Paris, 269 (1969) 858. E. M. P r a g e r and A. C. Wilson, J. Biol. Chem., 246 (1971) 5978. J. P. Locquet, J. S a i n t - B l a n c a r d a n d P. Joll~s, Biochim. Biophys. Acta, 167 (1968) 15o. N. Sharon, J. Jollbs and P. Joll~s, Bull. Soc. Chim. Biol., 48 (1966) 731. E. Maron, R. Arnon, M. Sela, J. P. P e r i n a n d P. Joll~s, Biochim. Biophys. Acta, 214 (197 o) 222. N. Arnheim, E. M. P r a g e r a n d A. C. Wilson, J. Biol. Chem., 244 (1969) 2085. E. M. P r a g e r a n d A. C. Wilson, J. Biol. Chem., 246 (1971) 7OLO. J. Haimovich, E. Hurwitz, N. Novik a n d M. Sela, Biochim. Biophys. Acta, 207 (197 o) 115. J. Haimovich, E. Hurwitz, N. Novik a n d M. Sela, Biochim. Biophys. Acta, 207 (197 o) 125. M. h n a n i s h i , N. Miyagawa, H. Fujio a n d T. A m a n o , Biken J., 12 (1969) 85. S. A. Barker, A. B. Foster, M. Stacey and J. M. W e b b e r , J. Chem. Soc., (1958) 2218. E. W. Thomas, Carbohydr. Res., 13 (197 o) 225. D. Shugar, Biochim. Biophys. Acta, 8 (1952) 302. E. Maron, C. Shiozawa, R. A r n o n a n d M. Sela, Biochemistry, IO (1971) 763. R. von Fellenberg a n d L. Levine, Immunochemistry, 4 (1967) 363 . W. J. Browne, A. C. T. North, D. C. Phillips, K. Brew, T. C. V a n a m a n and R. L. Hill, J. Mol. Biol., 42 (1969) 65. G. P. Hess a n d J. A. Rupley, Annu. Rev. Biochem., 4 ° (1971 ) lOl 3. T. C. V a n a m a n , K. Brew a n d R. L. Hill, J. Biol. Chem., 245 (197 o) 4583 . R. A r n o n a n d E. Maron, J. Mol. Biol., 51 (197 o) 7o3 .
Biochim. Biophys. A cta, 278 (i972) 243-249