topographic antigenic determinants recognized by monoclonal

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 6, Issue of March 25, pp. 3189-3198, 1982 Printed in U.S.A.

Topographic Antigenic Determinants Recognized by Monoclonal Antibodies to Sperm Whale Myoglobin*

(Received for publication, July 8, 1981, and in revised form, November 4, 1981)

Jay A. Berzofsky, Gail K. Buckenmeyer, Gloria Hicks, Frank R. N. Gurd, Richard J. Feldmann, and John Minna From the Metabolism Branch and the National Cancer Institute- Veterans Administration Medical Oncology Branch, National Cancer Institute and the Division of Computer Research and Technology, National Znstitutes of Health, Bethesda, Maryland 20205, the Washington Veterans Administration Medical Center, Washington, D. C. 20422, and the Department of Chemistry, Indiana University, Bloomington, Indiana 47405

Monoclonal antibodies of high affinity (-18’ “I) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, ie. topographic determinants. The third antibody distinguishes 140 Lys -+ Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu -+ Asp, in the antigenic site.

Until the advent of cell fusion techniques to generate hybrid tumor cell lines (hybridomas) producing monoclonal antibodies of any desired specificity (l), immunochemists had avail- able only 2 basic types of antibody as reagents or as primary subjects of research. Immune serum antibodies had the feature of desired overall specificity but were heterogeneous in both fine specificity and affinity. The heterogeneity of fine specificity could be minimized, although not eliminated, by affinity chromatography on fragments of the immunogen (2), but some heterogeneity of affinity remained. The other type of antibody, myeloma proteins (3), had the advantage of homogeneity but the disadvantage that they arose randomly, so that specificity could not be controlled and could not even be determined without large scale screening of antigens. Thus, the discovery of hybridoma technology created a revolution in immunochemistry, by bringing together the advantages of both systems without the disadvantages of either.

Most monoclonal antibodies produced to date have been

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

intended as reagents for use in biological research (4, 5). In a few cases, they have been used to study antibody diversity and amino acid sequence correlates of idiotopes (6, 7). Re- cently, monoclonal antibodies to defined protein antigens have been raised for the purpose of studying the antigenic sites on these antigens (8-10). In the case of myoglobin, a model protein with well defined primary and 3-dimensional structure, we have prepared high affinity (10’ M-’) monoclonal antibodies which manifest linear Scatchard plots and react with different antigenic determinants of sperm whale myoglobin (9). Besides studying the idiotypes of these,’ we have had 2 immunochemical goals: 1) to define the antigenic sites on the 3-dimensional surface of sperm whale myoglobin with which these interact, and 2) to determine the structure of the antibody combining sites, in order to understand the complementarity of structures of 2 globular proteins which have a high affinity for one another. The current report presents our findings on the frst of these goals.

Interestingly, several of the monoclonal antibodies react with topographic determinants, which we define as determinants consisting of residues far apart in the primary structure but brought together on the 3-dimensional surface of myoglobin by the folding of the native molecule. These topographic determinants must thus be conformation-specific. Almost all of the residues involved are outside of the previously reported sequential determinants described for goat and rabbit antisera (11). Moreover, the monoclonal antibodies can distinguish subtle changes in the fine structure of the antigenic determinants, such as the substitution of aspartic acid for glutamic acid.

At the completion of these studies we learned that comparable results had recently been obtained for monoclonal antibodies to human myoglobin by our colleagues at the Univer- sity of Melbourne.’ These are reported in the accompanying paper ( 10).

EXPERIMENTAL PROCEDURES

Monoclonal Antibodies-The preparation, subclass, affinity, and other initial characterization of the monoclonal anti-sperm whale myoglobin antibodies of clones 1 (HAL19-201-A10), 2 (HAL32-201- B l l ) , 4 (HAL39-201-C3), 5 (HAL43-201-Ell), and 6 (HAM1-201-F3) were described previously (9). Of these, all are yl K except clone 2, which is yzn K. All were made by fusing spleens of hyperimmunized ASW mice with NS1, a nonsecreting derivative of P3 X 63 plasma-

’ Y. Kohno, I. Berkower, G. Buckenmeyer, J. Minna, and J. A. Benofsky. Shared idiotopes among monoclonal antibodies to distinct determinants of sperm whale myoglobin, J. Immunol. (1982) 128, in press.

6th Annual Conference on Protein Structure and Function, Lorne, Australia, International Union of Biochemists Symposium 104, Feb- ruary, 1981.

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3 189

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3 190 Topographic Antigenic Determinants of Myoglobin

cytoma cell line. All had linear Scatchard plots with affinities between 0.71 and 2.2 X lo9 M” (9). Clone 3.4 was derived from the same fusion (designated “HAL”) as clones 1-5. However, the original “clone 3” (HAL38-200-E6) was found to manifest a biphasic Scatchard plot and to be inhibitable to only about 50% by certain myoglobins, whereas others, such as dwarf sperm whale, inhibited completely (data not shown). Therefore, it was subcloned by limiting dilution and subclones 3.4 (1D5) and 3.5 (2DI) obtained. Since the latter was inhibited completely by low concentrations of equine myoglobin, while the former was not inhibited at all, even at high concentrations, these were considered to represent the 2 clones in the original mixture. Clone 3.4, with affinity 0.2 X lo9 M”, is further characterized in the present report.

Myoglobins-The sperm whale myoglobin preparation, repurified by the method of Hapner et al. (12) from a commercial preparation (Biozyme, Batch 2, distributed by Accurate Chemical and Scientific, Hicksville, NY), was described previously (13). Equine myoglobin (Biozyme) was repurified by CM-Sephadex C-50 chromatography in phosphate buffer at pH 6.4 and a step gradient from ionic strength 0.01 to 0.1, and the major component was used.

Beef and sheep myoglobins were prepared from heart muscle by a modification of the method of Hapner et al. (12). The muscle, freed of most of the adipose tissue, was homogenized in 1.5 volumes of 70% saturated ammonium sulfate. After centrifugation at 11,800 X g for 1 h, the supernatant fluid was dialyzed against running tap water and centrifuged, and the supernatant was concentrated, dialyzed against phosphate buffer, pH 6.2, ionic strength 0.01, oxidized with a 2-fold excess of K3Fe(CN)6, and purified by CM-Sephadex C-50 chromatography using a step gradient of phospbate buffers of pH 6.2, ionic strength 0.01; pH 6.4, ionic strength 0.05; then pH 6.5, ionic strength 0.10. The major component, well separated from other peaks, was analyzed for the presence of low spin ferric denatured material by optical spectroscopy at pH 6.0 on a Cary Model 219 spectrophotom- eter, and the concentration was determined from the absorbance at 503 nm and from the absorbance maximum at 540 nm of the cyanide derivative (millimolar extinction coefficient 10.4).

Dog skeletal muscle myoglobin was purchased from Sigma (Lot 85C-8050). The preparations and sequencing of skeletal muscle myoglobins from dwarf sperm whale (14), dall porpoise (15), goosebeaked whale (16), minke whale (17), bottlenosed dolphin (18), killer whale (19), pilot whale (20), and California sea lion (21) were described previously. All were rechromatographed and re-ascertained to be free of denatured material by optical spectroscopy before use. Human heart myoglobin was a gift of Drs. Vincent Zurowski, John Hurrell, and Edgar Haber, Massachusetts General Hospital, Boston, MA.

Myoglobin Fragments-Fragments of sperm whale myoglobin prepared by cleavage with CNBr by the method of Marshall et al. (22) were described previously (23). Labeling of these with KI4CNO at the NHp-terminal a-NH2 group was also previously described (23).

Radioimmunoassay-The preparation of “H-labeled sperm whale

l.Op-*

0 0 8 80 8 0 0 8 o o o

FINAL CONCENTRATION OF SWMb FRAGMENT I 1 551 [“MI

FIG. 1. Competitive inhibition by myoglobin fragments of monoclonal antibody binding to radiolabeled sperm whale myoglobin. Final concentrations of the competitive fragment are shown on the abscissa. The ordinate is the bound/free ( B / F ) ratio for the labeled sperm whale myoglobin (7 m). The numbered curues correspond to the monoclonal antibodies of clones 1 through 6. The

myoglobin at the NH2-terminal wNH2 group, and the method of solution radioimmunoassay were described previously (9). Briefly, after attainment of equilibrium (in 0.04 M phosphate-buffered 0.15 M saline, pH 7.6, in the presence of 10 m g / d bovine y-globulin as carrier), polyethylene glycol of M, = 6000 to 7000 (final concentration IO%, w/w) was used to precipitate all of the immunoglobulin plus bound myoglobin, leaving the free myoglobin in solution. Bound and free labeled antigen were counted independently. Nonspecific binding by supernatants of P3 X 63 (the MOPC 21 myeloma protein-secreting parent of NS1) was subtracted from all bound antigen values.

For competition studies, at a constant concentration of labeled sperm whale myoglobin (7 n ~ ) , a dilution of each monoclonal antibody culture supernatant was determined which produced a bound/ free ratio close to 1.0, in the absence of inhibitor. Then bound/free ratios were determined in the presence of increasing concentrations of unlabeled competitive myoglobin, up to 40,000 n~ or over a 5,000- fold molar excess. The concentrations which resulted in 50% inhibition were taken as a measure of the relative affinities of the different myoglobins. However, these values cannot be taken as true dissociation constants, since terms involving the concentration of labeled sperm whale myoglobin and antibody enter into the derivation of the 50% inhibition concentration (see Ref. 24 and Footnote 2 of Ref. (9)).

RESULTS

Inability of the Monoclonal Antibodies to React with Frag- ments ofMyoglobin-In order to determine the fine specificity of the monoclonal antibodies raised against sperm whale myoglobin, we fist assessed their reactivity with fragments of the immunogen. To our surprise, none of them bound any I4C- labeled fragment (132-153) or fragments 1-55, even at large antibody excess, despite the fact that sera from the mice whose spleens were used for fusion bound both fragments (data not shown). To confirm these results a t large antigen excess, without the use of radiolabeled fragments, we assessed the ability of fragments 1-55 and 56-131 to inhibit the binding of tracer amounts of 3H-labeled sperm whale myoglobin (7 nM). Even at 8000 nM (1140-fold molar excess), we could not detect inhibition by either fragment (Fig. 1). Since the monoclonal antibodies, being homogeneous, should bind to a single site, a fragment which bound with an affinity within 103-fold of that of the native molecule should have shown complete inhibition in that range, and one with an affinity 104-fold lower than the native molecule should have shown partial inhibition. (For comparison, using conventional antisera, Hurrell et al. (25) found that fragment 132-153 bound with affinity only 100-fold lower than the native myoglobin.) These results

.5 .5

0 8 80 8 0 0 8 O O o

FINAL CONCENTRATION OF SWMb FRAGMENT 156-1311 [ n M ]

final dilutions of hybridoma culture supernatant used were A and B, clone 1, 1:80 (3.9 n~ in binding sites); clone 2, 1:40 (4.2 nM); clone 3, 1:20 (13 nM); clone 4, 1:40 (6.2 nM); clone 5, 1:40 (5.6 n ~ ) ; clone 6, 1:5 (10 nM) In C and D, the dilutions were the same except clone 3.4, for which a new supernatant was used at 1:5 dilution (approximately 12 nM in binding sites).


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Topographic Antigenic Determinants of Myoglobin 3191

suggested that the monoclonal antibodies were specific for structural features present only on the native protein but not on any of the fragments (see “Discussion”).

Mapping of Antigenic Residues Using Species Variants of Myoglobin-Since the monoclonal antibodies did not bind even relatively large cyanogen bromide cleavage fragments, we had to use myoglobins with native tertiary structure and known amino acid substitutions to determine which residues were critical for binding. The sequences of the myoglobins used are shown in Table I.

In order to use these myoglobins to define antigenic residues, the following assumptions were necessary: 1) a given homogeneous monoclonal antibody can bind to only one unique antigenic site (determinant) on the antigen molecule; 2) the gross tertiary structure of myoglobins from all mammals is very similar. (This assumption has proven true for those myoglobins studied by x-ray crystallography (33-37). In fact, there is considerable conservation of structure in molecules as far removed as plant leghemoglobins (38).) 3) Therefore, amino acid substitutions which affect binding by a monoclonal antibody are more likely within the antigenic site than far from it; 4) the lower the affinity of a myoglobin species variant for a given monoclonal antibody, the greater must be the extent of changes in residues within the antigenic site bound by that antibody, relative to the immunogen.

Based on these assumptions, we devised the following strategy to analyze the fine specificity from binding affinities and sequence differences: 1) determine the relative affinities of a number of mammalian myoglobins for the antibody; 2) search for residues at which the low affinity myoglobins differ from the immunogen (sperm whale myoglobin) and all of the high affinity myoglobins; 3) assess whether there is a unique difference shared by all the low affinity myoglobins but none of the high affinity myoglobins; if so, this is a prime candidate to be part of the site; and 4) if there is no single substitution that can account for the lower affinity of all the low affinity myoglobins, assess whether there is a group of substitutions

close together in the 3-dimensional structure of myoglobin which in aggregate could account for all of the low affinity myoglobins. Using this approach, we have been able to define antigenic residues for 3 of the monoclonal antibodies with little or no ambiguity. The number of myoglobins tested is so far insufficient to unambiguously identify residues recognized by the other 3 antibodies.

A Topographic Site Involving Glu 4-Lys 79 Recognized by Clone 3.4 Monoclonal Antibody-The relative affinities of a series of myoglobins for antibody from clone 3.4 were determined by competitive binding radioimmunoassay (Fig. 2). As discussed under ‘‘Experimental Procedures,” the concentration of competitor resulting in a 50% reduction in the bound/ free ratio for labeled sperm whale myoglobin could be taken as an estimate of the dissociation constant. Although other

8 Mmke Whale Mh s Bonlenosed OOlUhin Mb

A Dall Porpo~se Mh

FIG. 2. competitive^ inhibition by various myoglobins of clone 3.4 antibody binding to 3H-sperm whale myoglobin. For- mat is the same as in Fig. 1. Final concentration of labeled sperm whale myoglobin, 7 nM. Final concentration of monoclonal antibody, approximately 12 nM in binding sites. Variation in the initial bound/ free (B /F) value in the absence of inhibitor was due to small variation in the antibody concentration in different experiments, since it was impossible to test all of the myoglobins (Mb) simultaneously. Any effect of these variations on the relative affinity measurement is negligible compared to the differences in affinity considered in the analysis.

TABLE I Amino acid sequences of myoglobin

Only positions at which substitutions occur with respect to sperm whale myoglobin are listed. A dash indicates that the residue in question is identical with that in sperm whale myoglobin. The residue numbers are read vertically. The conventional one-letter code for amino acid residues is used: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

Residue Number 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Myoglobin 1 4 5 0 9 2 3 5 9 1 2 7 0 4 5 1 5 1 3 4 7 6 7 4 3 6 0 1 5 9 1 9 0 3 6 7 0 0 1 2 4 7 8 9 2 0 2 4 5 7 8 ~ 2 1 1 1 1 2 2 2 2 3 3 4 4 5 5 5 5 6 6 7 0 0 0 9 9 9 ~ ~ 1 1 1 1 1 2 2 2 2 2 2 2 3 4 4 4 4 4 4 5 5

S p e n Whale V E G P L H V A A V A D I K S E R T A E A V T A E L P Q T I I E A H H S R P G D G A Q G N K I A K K E Y Q 26,27

Dwarf Sperm Whale - - - - - - - - - I - - - - H - - S - - - - - - - - - - - - - - - - - - - - A - - - - - S - - - - - - - - 14

Dall Porpoise G - - - - N - G - L - - V - G - K - - - - N - G D - - - - - - - - - - - - - A E - - - - - - - T - - - F H 15

Reference

GoosebeakedWhale G - A - - - - - - L S E - - G - K S - - - H - G - - - - - - - D - - - - - - S - - - - A T - - - - - - F H 16

BottlenosedDolphin G D - - - N - G - L - - V - G - K - - D - N - - D - - - - - - - - - - - - - A E - - - - - - - - - - - F H 18

MI nke Whale - D A H - N I - - - - - - - G - K - - - - N - G - - - - - - - D - - - - - - A E ” - A - - ” ” - F - 17

Pilot Whale G D - - - N - G - L - - - - G - K - - D - N - - - - - - - - - - - - - - - - A E - - - - - - - - - - - F H 20

Killer Whale G D - - - N - G - L - - - - G - K - - D - N - - D - - - - - - - - - - - - - - - - A E - - - - - - - - - - - F H 19

CalfforniaSeaLion G D - - - N I G - L V E V - G - K S D - R K - G D - - - - - - - - - Q - K - - - - T H A K N - - - R - F - 21

Human G D - - - N - G - I P E v - G - K s D - - A - G - I - - - - V - C Q Q - K - - - - - - - - - M S N - - F - 28

Dog

Horse

Beef G D - - A N A G - - - E V T G - K - - - - N - G - V H E N V - D - - - A K - S N A - - - S N A E - - V F H 31

Sheep G O - - - N A G - - - E V T G - K - - - - N - G - V H E N - V D - - - A K - S N - - - - S N M - 0 - V F - 32


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3192 Topographic Antigenic Determinants of Myoglobin

PLATE 1. Computer-generated space-filling molecular models of the 3-dimensional structure of several myoglobins, indicating the topographic structure of antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin. The stereo-pairs may be viewed in 3 dimensions using an inex- pensive stereoviewer such as the “stereoscope” sold by the Hubbard Scientific Co., Northbrook, IL. The amino acid side chain functional groups are color-coded by type, to demonstrate interactions among these. Primary amino groups (Lys, k g , NHn-terminal), dark blue; amide nitrogens (of Asn and Gln) and ring nitrogens of His, light blue; carboxylic acid oxygens (Glu, Asp), dark red; amide oxygens (Asn and Gln), hydroxyl oxygens (Ser, Thr, Tyr), and sulfur atoms (Cys, Met), pink; aromatic ring carbons (Phe, Tyr, Trp, His, and the heme porphyrin ring including side chains), green; and aliphatic residues (Gly, Ala, Leu, ne, Val), yellow. The peptide backbone and the aliphatic portions of residues with other functional groups (eg. the @-E carbons of Lys) are shown in white. The 3-dimensional structure

of sperm whale myoglobin is taken directly from the x-ray crystallographic coordinates (33, 34). The structures of the other myoglobins were generated from that of sperm whale myoglobin by substitution of the known amino acid replacements followed by rotation of the side chain to minimize any unfavorable van der Waals contacts, using a computer program recently described (39). Since no bad van der Waals contacts were encountered and since the x-ray crystallographic structures of the several myoglobins studied are virtually identical with that of sperm whale (35-37), these are reasonable models of the substituted myoglobins. The computer methods used to generate the stereoscopic images of space-filling models have been described (40, 41). The “front view” is arbitrary. Other views are relative to this one. Stereo views: A, sperm whale myoglobin, front; E , killer whale myoglobin, front; C, sperm whale myoglobin, bottom; D, dall porpoise myoglobin, bottom; E, sperm whale myoglobin, left; F, killer whale myoglobin, left; G, bovine myoglobin, left; H, horse myoglobin, left.


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QUI

PLATE 1 continued

terms affect this value (9, 24), with all other parameters held constant, we can compare relative affinities by this method.

Using the strategy outlined above, we found that one and only one substitution could account for all of the low affinity myoglobins. All of the high affinity myoglobins share Glu 4 with the immunogen, whereas all of the low affinity myoglobins have an Asp substitution at this position (Table 11). Examination of the 3-dimensional structure of sperm whale myoglobin reveals that Glu 4 forms a close ionic bond, or salt bridge, with the eNH2 group of Lys 79 (Plate L4). The ion pair between residues 4 and 79 is fully conserved among mammalian myoglobins, which has been taken as evidence for the importance of this interaction in stabilizing the native structure by connecting the A helix with the EF corner (42). From the crystallographic coordinates, the carboxyl oxygen to amino nitrogen distance is only about 1.8 A (34). In contrast,

when the Glu 4 is replaced by an Asp residue, as in killer whale myoglobin (Plate lB), the distance of closest approach is only 3.2 A, allowing only a somewhat weaker bond. Thus, we postulate that the monoclonal antibody recognizes the Glu 4-Lys 79 couple and that, when Glu is replaced by Asp, the comparatively unneutralized charge of the Asp lowers the affinity. As indicated in Plate 1, A-D, the detailed topography of the site is altered by this substitution, in addition to the decrease in stability resulting from the weakening of the ion pair interaction. Since these 2 residues, Glu 4 and Lys 79, are far apart in the primary structure but are brought together by the folding of the native protein molecule, this antigenic determinant depends for its existence on the native conformation. Therefore, it is appropriate to designate it as a topographic determinant. Moreover, none of the CNBr cleavage fragments contains the complete determinant, so it is not


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TABLE I1 Inhibition of monoclonal antibody 3.4 binding by myoglobin

variants Concentration for

50% I n h i b i t i o n I n h i b i t o r Myoglobin 1 4 5 8 9 1 2 nM

Sperm Whale

Dwarf S p e n Whale

Dall Porpoise

Goosebeaked Whale

Bottlenosed Dolphin

Minke Whale

K i l l e r Whale

C a l i f o r n i a Sea Lion

Human

Dog

Horse

Beef

V

G

G

G

G

G

G

G

G

G

8

46

333

10

8.000

>40,000

>40,000

3,200

>X300

>40,000

>40,000

>40,000

surprising that none of them binds to clone 3.4 antibody. Since Lys 79 is invariant, there are no natural myoglobin variants with which we can further test the role of this residue in the antigenic determinant.

One second order effect may allow us to discern the involve- ment of a third residue, histidine 12. Of the high affinity myoglobins, dall porpoise myoglobin shows the lowest affinity, by a whole order of magnitude (Table 11). This is the only one of the high affinity myoglobins which has the His 12 of sperm whale replaced by an asparagine (Plate 1, C and D). The His ring can come within 9.4 A of the Glu residue at position 4, as determined from computer analysis of the x-ray coordinates. While we do not expect any interaction between these residues at this distance, they are close enough to be part of a single antigenic determinant.3 Because of the high degree of exposure to solvent of the His 12 imidazole ring, a strong electrostatic interaction with other residues such as those as positions 4 or 79 is unlikely (44-46) and is ruled out at the pH of our antibody binding measurements at which His 12 will be virtually completely in the uncharged form.

A Topographic Determinant Recognized by Clone 1 Mon- oclonal Antibody-The relative affinities of a series of myoglobins for clone 1 antibody were determined from the competitive binding curves (Fig. 3). The affinities fal l into 2 groups, those comparable to that for sperm whale myoglobin and those one to two orders of magnitude lower (Table 111). Unlike the situation for clone 3.4 antibody, no single amino acid substitution is present in all of the low affinity myoglobins and none of the high affinity myoglobins, which could account for the observed differences in affinity (Tables I and 111). Therefore, we searched for groups of substitutions close enough on the 3-dimensional surface of the native myoglobin to be part of the same determinant, which together could account for all of the low affinity myoglobins. The only such group which fulfilled these criteria consisted of residues 83 and 144 plus 145. The high affinity myoglobins have substitutions at none of these positions. Of the low affinity myoglobins, killer whale and California sea lion have 83 Glu + Asp but no change at 144 or 145; human and beef have 144 Ala + Ser and Glu, respectively, but no substitution at position 83; human and sheep also have an amide substituted for Lys 145 but no change a t position 83, and dall porpoise, otherwise

Complementarity-determining residues of antibodies may span 20 A to even 30 to 40 A (D. R. Davies, personal communication, and references reviewed in Refs. 24 and 43).

a California Sea Lion Mb

o Equine Mb

n Dall Porpo~se Mb Human Heart Mb

Killer Whale Mb

rn GoosRbeaked Whale Mb

2.0 r

BiF 1.0

0 8 80 8M) 8Mx) 4o.m FINAL CONCENTRATION OF INHIBITOR [nM]

FIG. 3. Competitive inhibition by various myoglobins of clone 1 monoclonal antibody binding to 3H-sperm whale myoglobin. Format and protocol are the same as in Figs. 1 and 2. Final concentration of labeled sperm whale myoglobin, 7 m, and of clone 1 monoclonal antibody, approximately 4 m in binding sites. B/F, bound/free; Mb, myoglobin.

TABLE 111 Inhibition of monoclonal antibody 1 binding by myoglobin variants

1 1 1 1 1 1 1 1 1 1 1 Concentrat ion for

Inhibi tor Myoglobin 3 6 8 1 9 0 2 0 2 4 5 7 8 1 2 8 8 8 9 0 1 3 4 4 4 4 4 4 5 5 50% I n h i b i t i o n

nM

Sperm Whale

Dwarf Sperm Whale

Goosebeaked Whale

Dog

Horse

P i l o t Whale

K i l l e r Whale

C a l i f o r n i a Sea L i o n

Human

Dall Porpoise

Beef

Sheep

I

E Y Q

_ " - F H

- F -

- F -

- F H

- F H

- F -

- F -

- F H

V F H

V F -

10

13

3

5

3

24

240

160

470

500

>40,000

>40,000

very similar to sperm whale, has changes at both positions, 83 Glu + Asp and 144 Ala + Thr (Table 111). Since either a change at residue 83 or a change at residues 144 or 145 without the other lowers the affinity, we believe that both sets must be involved in the antigenic determinant (Plate 1, E-G).

Strong confirmation for the importance of the 83 Glu 4

Asp substitution comes from a comparison of myoglobins from killer whale and pilot whale. These differ from each other at only a single position in the entire sequence, namely position 83. Yet pilot whale myoglobin, with 83 Glu, falls in the high affinity group (24 nM), while killer whale myoglobin, with 83 Asp, has a 10-fold lower affinity (240 nM) (Table 111 and Plate IF). Unfortunately, we do not have a similar pair of myoglobins which differ only at position 144 or 145. However, of the 3 low affinity myoglobins with substitutions at 144, beef, which has the least conservative change (Ala --* Glu), has by far the lowest affinity, even though it has no change at position 83.4

Beef myoglobin also bas a substitution of Leu for Val at position 86. This residue is tucked back deep in the crevice between residues 83 and 144/145 and probably is not accessible to antibody. Moreover, examination of three-dimensional models suggests that this substitution does not significantly affect the topography of the other residues, such as 83, 144, and 145, on the surface. Therefore, we believe that it is the very nonconservative substitution of Glu for Ala at position 144, rather than the Leu + Val substitution at position 86, which accounts for the failure of beef myoglobin to show any detectable affinity for clone 1 antibody. The same argument applies to residue 145 in sheep myoglobin.


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Topographic Antigenic De

The same point applies to sheep, which has a basic residue at position 145 replaced with a neutral one. Therefore, we believe all 3 residues are involved in this determinant."

Computer analyses demonstrate that the minimum distance between Glu 83 (carboxyl oxygen) and Ala 144 (p carbon) is 11.7 A (see Plate 1E). They are separated by a crevice in the surface of the molecule. In contrast, Asp 83 (carboxyl oxygen) can reach to within 9.2 A of the Ala 144 p carbon in killer whale myoglobin (Plate 13'). Based on the x-ray crystallographic coordinates (33, 34), the distance between Glu 83 (carboxyl oxygen) and Lys 145 (€-amino nitrogen) is 7.1 A. If the orientation of the a-P bond is unchanged, substitution of Asp for Glu a t position 83 reduces this distance to 5.7 A. If, however, the side chains of both residues 83 and 145 are rotated in a computer simulation to minimize this distance without creating any unfavorable contacts with other residues, Glu 83 could reach to within 3.2 A of Lys 145, while Asp 83 could reach to within 3.4 A. While these distances suggest that an electrostatic interaction between these oppositely charged residues is possible, we have no evidence to indicate that such an interaction actually occurs. The Glu -+ Asp substitution causes very little difference in the myoglobin charge array that has been examined in detail for myoglobins of the following species: sperm whale, dwarf sperm whale, sei whale, minke whale, humpback whale, Amazon River dolphin, common porpoise, pilot whale, and bottlenosed dolphin (44, 45). Therefore, it is more likely that the substitution Glu +

Asp changes the topographical features recognized by the monoclonal antibody (Plate 1, E and F) in the complex. I t is also possible that the substitution of residue 83 narrows the crevice separating that residue from residues 144 and 145 sufficiently to lower the antibody binding affinity, if this is a feature complementary to the antibody (see "Discussion"), The substitutions at position 144 may result in packing defects in the antibody-antigen complex. The Ala -+ Glu change in bovine myoglobin could contribute destabilization, moreover, by alteration of the surface charge array (Plate 1G). In addition, we should consider the observation that Glu 83 (carboxyl oxygen) in sperm whale myoglobin can reach to within 3.2 A of a ring nitrogen of His 81, close enough for a hydrogen bond, whereas the carboxyl oxygens of Asp 83 in killer whale or dall porpoise myoglobins cannot get closer than 6.8 A from this His 81 nitrogen (Plate 1, E and F). The pK, measured for His 81 by proton NMR measurements is 6.31 for both pilot whale, Glu 83, and dall porpoise, Asp 83, at an ionic strength of 0.1 M (47). Hence, the fractional charge of His 81 appears to be unaffected by the amino acid substitution at position 83. Under the test conditions, the side chain of His 81 will be substantially uncharged. Nevertheless, in the antigen-monoclonal antibody complex, the position of the side chain of residue 83 relative to His 81 may be important.

Identification of Lys 140 as a Critical Residue in the Antigenic Determinant Recognized by Clone 5 Antibody-A similar analysis of the competitive binding curves for monoclonal antibody of clone 5 (Fig. 4 and Table IV) results in a striking dichotomy between 7 myoglobins which inhibit at low concentration and 5 which either show no inhibition or inhibit only a t concentrations several thousandfold higher than required for sperm whale myoglobin (Table IV). In this case, a single amino acid substitution, Lys + Asn a t position 140, is the only one which could account for all the low affinity

'' Our recent finding that rabbit myoglobin (which has a Gln substitution at position 145 but no substitutions at positions 83, 144, or 148) has an affinity nearly as low as that of beef and sheep myoglobins (>7000 nM required to produce 50% inhibition) confirms the assign- ment of residue 145 and makes it unlikely that the substitution at position 148 accounts for the low affinity of beef and sheep myoglobins.

terminants of Myoglobin 3195

2'o i D Callfornu Sea Llon Mb

~ EqulneMb

3 Dall Porpovse Mb

Human HeaR Mb

EiF - 8 Bo 8w3 8ooo 4o.ooo

FINAL CONCENTRATION OF INHIBITOR lnM]

FIG. 4. Competitive inhibition by various myoglobins of clone 5 monoclonal antibody binding to 3H-sperm whale myoglobin. Format and protocol are the same as in Figs. 1 and 2. Final concentration of labeled sperm whale myoglobin, 7 nM, and of clone 5 monoclonal antibody, approximately 5 nM in binding sites. B/F, bound/free; Mb, myoglobin.

TABLE IV Inhibition of monoclonal antibody 5 binding b y myoglobin UariURts

1 1 1 1 1 1 1 1 1 1 1 Concentrat ion for 2 2 2 3 4 4 4 4 4 4 5

Inhibi tor Myoglobin 7 8 9 2 0 2 4 5 7 8 1 50% I n h i b i t i o n

m

7

Sperm Whale

Human

32 Goosebeaked Uhale

10 Dall Porpoise

- - - - - - 26 - Dwarf Sperm Whale - - - 5

A Q G N K I A K K E Y 17

- - - R - F >20,000 N C a l i f o r n i a Sea L i o n T H A K

F 55 K i l l e r Whale

21 Minke Whale

M S N - - F 30 " _ "

Dog T E A K N - - - - - F >40,000

6400

" " - - T - - - F

- - A T " - - - - F

- - A " " - - - F

"""""

Horse

Beef

Sheep

" - T N " - - - F

" _ S N M - a - V F >40,000 " _ S N A E - - V F >40,000

- myoglobins but does not occur in any high affinity myoblobin (Plate 1, E and H). Therefore, we can safely identify Lys 140 as a critical residue in the determinant recognized by clone 5 antibody. Since replacement of this basic residue by a neutral one results in such a drastic decrease in affinity, it is probable that a significant part of the binding energy is contributed by the interaction of an acidic residue in the antibody binding site with the E - N H ~ group of Lys 140. Note also that the affinity of clone 5 antibody is not affected by substitutions at positions 144 or 145 which affect the affinity of clone 1 antibody, which in turn is not affected by substitutions at position 140 (Tables I11 and IV). Thus, even though the determinants recognized by these 2 monoclonal antibodies are close together, they appear not to overlap. This prediction has been confirmed by the demonstration of simultaneous binding of the 2 antibodies to myoglobin.' Also, this observation supports our assumption that these substitutions affect binding directly rather than through propagated conformational changes.

DISCUSSION

We have identified, by use of myoglobin sequence variants, critical antigenic residues involved in the binding of 3 mouse monoclonal hybridoma antibodies to native sperm whale myoglobin. These have allowed us to detect, in 2 of the cases, topographic antigenic determinants involving residues far apart in the amino acid sequence but brought together on the


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surface of the native protein molecule, namely Glu 4-Lys 79 and Glu 83-Ala 144-Lys 145. A third monoclonal antibody recognizes a determinant including Lys 140. Although we have not yet identified a second residue recognized by this monoclonal antibody, the antibody must be conformation-specific since it does not bind any of the CNBr cleavage fragments of myoglobin. Independent evidence for such topographic determinants was obtained in experiments performed concurrently with ours and reported in the accompanying paper by East et al. (10). These investigators have identified 2 similar topographic determinants recognized by a monoclonal antibody to human myoglobin. These determinants also appear to involve residues far apart in the sequence but brought together by the tertiary structure of the native molecule. The critical residues 87 and 142 recognized by one of these antibodies are close, in fact, to the ones recognized by our clone 1 antibody.

Previous studies of antigenic sites on sperm whale myoglobin have relied heavily on synthetic peptides (11, 48). In particular, 5 regions of 6 to 8 consecutive amino acid residues in the sequence were reported to be the sole antigenic determinants on sperm whale myoglobin (11). These studies used immune antisera raised in several goats and rabbits. Some of these sites have been confirmed by others (48). However, because we have been unable to detect antigenicity for 2 of these synthetic sites (residues 56 to 63 and residues 93 to 102) using both early and hyperimmune antisera from 2 goats, a sheep, and mice of several high responder strains, both by direct binding and by competition,6 we decided to analyze our data de novo, without employing any assumptions about these previously reported sequential determinants. This was espe- cially important since none of our monoclonal antibodies bound to any of the 3 cyanogen bromide cleavage fragments, which together constitute the whole sequence of myoglobin (except for the cleavage points) and contain all 5 of the previously reported antigenic sites. If our monoclonal antibodies were specific for any of these 5 sites, they would have been expected to bind to one of the cyanogen bromide fragments. Moreover, the determinants that we and East et al. (10) detected turned out to differ from those reported (11) both in their topographic character and in most of their amino acid composition. Indeed, most of the residues were outside the limits of the nearest neighbor analysis for these sites (49). Therefore, there clearly exist other antigenic determinants on sperm whale myoglobin not encompassed by the antigenic structure reported by Atassi (11).

Three interpretations of the failure of the monoclonal antibodies to bind fragments were possible. Either the mono- clonals all happened to bind to determinants exactly spanning the CNBr cleavage sites, a statistically unlikely possibility, or they all were extremely specific for the native conformation of determinants for which the appropriate sequence was actually represented intact in a fragment, but in the wrong conformation, or they recognized determinants consisting of residues on different fragments, widely separated in the sequence, but brought into close proximity by the folding of the native molecule. As we have shown, the specificity of a t least 2 of the monoclonal antibodies can be explained by the last of these situations, although the other 2 cases may occur as well. The data so far on the antibody of clone 5 are compatible with either of the latter 2 explanations. Since 6 out of 6 monoclonal antibodies tested failed to react with any of the fragments of myoglobin, we would expect this type of antibody specific for the native conformation of myoglobin to occm with high frequency in immune serum antibodies as well. On the one hand, Atassi and co-workers (50) report the ability to absorb all of the antimyoglobin antibodies on affinity columns of 5

J. A. Benofsky and D. J. Killion, unpublished observations.

synthetic peptides of 6 to 8 residues each. Antibodies of the type represented by our monoclonal antibodies would not be expected to bind to these columns. On the other hand, when rabbit, goat, and sheep serum anti-myoglobin antibodies were passed exhaustively over 3 affinity columns each bearing one of the 3 cyanogen bromide cleavage fragments encompassing the whole sequence of myoglobin, 30 to 40% of the antibodies in each serum did not bind to any of the fragments even though they had a high affinity for the native m~lecule .~ This result is consistent with the notion that a significant fraction of immune serum antibodies is directed a t topographic determinants for which all residues are present only in the native molecule. Our monoclonal antibodies presumably belong to this subset.

There is considerable precedent in the literature on conventional antisera for conformational determinants on proteins such as myoglobin (51, 52). Also, evidence suggestive of possible topographic determinants exists for lysozyme (53), insu- lin (54), tobacco mosaic virus (55), cytochrome c (56), and fetal hemoglobin (57). For instance, goat antisera to sperm whale myoglobin were not a t all inhibited by beef myoglobin, even though these share the reported determinant involving residues 56 to 62 (58). Similarly, goat antisera to sperm whale myoglobin, fractionated on an affinity column of fragments 56-131, showed marked differences in affinity for horse and pig myoglobins despite the fact that these are identical in sequence in 2 of the 3 reported sites on this fragment (residue 56 to 62 and 94 to 99) (58). Using a similar approach, East et al. (59) showed that conventional antisera to beef myoglobin, fractionated on fragments 1-55 bound to Sepharose in order to isolate those antibodies specific for this part of the molecule, showed very different affinities for beef, sheep, and pig myoglobins, despite the fact that these proteins are identical in the sequence 15 to 22, which had been reported to be the sole antigenic determinant in this part of the molecule (11). More- over, East et al. (59) found 2 subpopulations of antibodies to this region of the molecule, one cross-reacting with sheep myoglobin with a much higher affinity than the other. There- fore, in all of these cases, if antibodies do bind these linear sequences, the sequences alone must not be the complete determinants. Rather, other residues nearby on the 3-dimensional surface must be involved in antibody binding. As pointed out by Hurrell et al. (58), the large differences in affinity cannot be attributed to subtle differences in conformation of sequential determinants, since these myoglobins have the same overall tertiary structure, while the differences in affinity approach those observed for isolated fragments compared to native myoglobins. These results all suggest probable topographic determinants. Similar results for other proteins have been reviewed by White et al. (60), who point out that about 80% of evolutionary amino acid substitutions are immunologically detectable, a finding which cannot be reconciled with the view that only about 15% of the residues participate in antigenic sites.

These results together with ours are consistent with the hypothesis that there are topographic regions, or domains, on the surface of a protein, which are antigenic (58,59). Different overlapping portions of a domain may be recognized by different antibodies, which therefore have related, but not identical, specificity. For instance, residues 83 and 144 of sperm whale myoglobin recognized by our antibody of clone 1 and residues 87 and 142 of human myoglobin recognized by the antibody of East et al. (10) may be part of a larger domain. Another one of our monoclonal antibodies (clone 2) to sperm wbole myoglobin also appears, from preliminary observations, to recognize residue 144, but not residue 83 (data not shown). ’ G. Lando and M. Reichlin, personal communication.


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These 3 antibodies therefore recognize overlapping but not identical determinants within a domain. Of course, the approach we have used, employing native myoglobins from different species, can define only residues for which known substitutions exist. Residues conserved in all the mammalian myoglobins studied could be part of an antigenic domain, but we would not detect them by this technique. In order to extend these studies to define other nearby surface residues involved in the sites recognized by the antibodies described, we are currently undertaking to prepare semisynthetic myoglobins with single amino acid substitutions and native tertiary structure. These should be powerful tools to define more completely these antigenic determinants and domains. In addition, electrostatic potential energy maps offer a tool to study the perturbations in the surface topography that will occur as a result of subtle alterations in the charge array of the protein.*

It is striking that 2 of the substitutions at critical positions which destroy antigenicity are Glu ”+ Asp substitutions. We have recently found a Glu + Asp and an Asp -+ Glu substitution which can diminish the antigenicity of myoglobins for immune T lymphocytes (including a possible topographic site involving residues brought together by the folding of the native mole~u le ) .~ Since the carboxylic acid functional groups of these amino acids are the same, it was remarkable that such conservative substitutions should have such profound effects on binding affinity. Rather than postulate that the difference in length of one methylene group affects exclusively the ability of the carboxyl group to interact with a contact residue on the antibody, we believe one explanation is illustrated by the case of the monoclonal antibody of clone 3.4. In that case, Glu 4 formed a much stronger ionic bond with Lys 79 than could Asp 4. Thus, the electrostatic potential field around the carboxyl group was perturbed by the proximity to another residue in the antigenic site, which in turn depended on side chain length. It is also possible that Glu + Asp substitutions may cause small local conformational changes, since Glu favors a-helical structure more than does Asp (61). Another example of a localized conformational change that could be caused by the substitution of Asp for Glu at position 4 might be a slight movement of the whole A helix, the orientation of which is stabilized by the interaction of residue 4 with Lys 79.

Another aspect of the topographical effect of the Glu 3

Asp substitution is in the degree of exposure of the carboxyl group to the solvent (62, 63). The shorter Asp side chain allows less exposure to the solvent than for a Glu side chain in the same residue positions. In fact, Asp 4 is exposed approximately half as much as Glu 4, and Asp 83 is exposed approximately 10% less than Glu 83 in the native myoglobin structure (44-46). If the acidic side chain is a recognition site with either Glu or Asp at that position, the free energy change due to the binding will be greater for the case of Glu because it can undergo a greater change in solvent accessibility by virtue of the combination with the antibody binding site, and SO contribute more significantly to the electrostatic free energy change accompanying binding. Similar effects have been recognized for several charged groups involved in stabilizing the assembly of hemoglobin subunits, another example of protein- protein interaction in which electrostatic interactions appear to be significant (64-67). Possible entropic effects of solvent accessibility changes would be expected to contribute to the binding as well.

The 3 examples of charge interactions illustrated by the

J. B. Matthew, personal communication. I. Berkower, F. R. N. Gurd, and J. A. Berzofsky, manuscript in

preparation.

apparent monoclonal antibody specificities are in interesting contrast. That involving residue 83 has been discussed in terms of the interaction of an anionic group on the myoglobin with a putative cationic group in the antibody binding site. Close complementation with exclusion of solvent would pro- mote strong electrostatic and, quite probably, hydrogen bond- ing interactions (42, 66-70). The example involving residue 4 requires a complementary array on the antibody site probably involving a pair of charged groups to interact with the 4-79 couple in myoglobin, probably in a stable quadrupolar interaction. The example involving residue 140 appears to be one in which complementarity requires the presence of a cationic side chain at that location. If such a positive charge is lost to a neutral substitution, the putative anionic group in the antibody combining site may become buried without pairing with an oppositely charged group, an arrangement that would be markedly unfavorable. These monoclonal antibodies allow delineation of protein antigenic determinants with much greater resolution than would be possible with conventional serum antibodies.

Now that we can define critical residues on the surface of myoglobin which are involved in binding to these monoclonal antibodies, we are trying to identify the specific contact residues in the antibody combining sites which interact with these. We hope to employ these monoclonal antibodies as models to understand the nature of mutually complementary sites on two globular proteins (antigen and antibody) which have high affinity for one another. These studies should extend those in which the model antigen was a small hapten which could bind to a myeloma protein (3). One difference on which we can speculate already is the overall shape of the antibody combining site. The large concavity between the residues 83 and 144 recognized by clone 1 antibody suggests that the complementary antibody combining site may be convex. The use of small haptens to study antibody combining sites naturally favored the detection of combining sites which were cavities or crevices within the antibody molecule (43). Protein antigens may allow detection of antibody combining sites of completely different topography.1o

Acknowledgments-We thank Drs. Vincent Zurowski, John Hur- rell, and Edgar Haber for their gift of human myoglobin, Dr. Morris Reichlin for the gift of beef myoglobin used in the early experiments, Margaret A. Flanagan for assistance in the electrostatic analysis, Mark Busch and Dr. Joseph L. Meuth for assistance in preparation of some of the myoglobins, and Drs. Sydney J. Leach, Moms Reichlin, David Sachs, and Alan Schechter for critical reading of the manuscript.

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J A Berzofsky, G K Buckenmeyer, G Hicks, F R Gurd, R J Feldmann and J Minnasperm whale myoglobin.

Topographic antigenic determinants recognized by monoclonal antibodies to

1982, 257:3189-3198.J. Biol. Chem.

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