immunogenicity of synthetic peptides corresponding to flexible … · 2001-06-23 · segments of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 18, Issue of June 25, pp. 10513-10519,1389 Printed in U.S.A.

Immunogenicity of Synthetic Peptides Corresponding to Flexible and Antibody-accessible Segments of Mouse Lactate Dehydrogenase (LDH)-C4*

(Received for publication, January 9, 1989)

Holly H. HogrefeS, Pravin T. P. Kaumaya, and Erwin Goldberg8 From the Department of Biochemistry, Molecular Biology & Cell Biology, Northruestern Uniuersity, Euanston, Illinois 60208

The immunological properties of a panel of synthetic peptides that represent the most accessible and mobile segments of the lactate dehydrogenase (LDH)-C4 molecule were characterized. Peptides corresponding to mouse LDH-C4 amino acid sequences: 1-14b, 5-15,

316, and 318-330 were synthesized and compared in terms of binding antibodies raised in rabbits against the intact protein. Six of these sequences were cova- lently coupled to diphtheria toxoid and used to immu- nize groups of rabbits. LDH-Co-specific antibodies were detectable in immune sera by enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation assays. The immunogenicity of the mouse LDH-C4 peptides in rabbits could be ranked in the following order: 5-15,304-316 z 211-220,274-286 > 49-58, 97-110. The immunological properties of these short synthetic peptides did not correlate with features of the mouse LDH-C4 structure except that the most active sequences appeared to be those that dif- fered from the somatic isozymes to the greatest extent. These results have direct bearing on the selection of immunogenic LDH-C4 peptides for contraceptive vaccine studies in humans and non-human model systems.

49-58,97-110, 211-220, 231-243, 274-286, 304-

~ ~~~~~~ ~~~~~~~~ ~ ~

In an attempt to develop an immunocontraceptive vaccine (Goldberg et al., 1983), the antigenic structure of the testes- specific isozyme of lactate dehydrogenase, LDH‘-C4, has been studied (Wheat and Goldberg, 1985). This isozyme is expressed only by spermatogenic cells (Goldberg, 1977) and has been found on the surface of mature spermatozoa (Erickson et al., 1975). The numerous amino acid sequence differences between the testicular and somatic LDH isozymes (-70% homology) (Eventoff et al., 1977; Li et al., 1983; Millan et al., 1987) render them antigenically distinct (Goldberg, 1971; Liang et al., 1986). Immunization of female animals with LDH-G purified from mouse testes results in the production of highly specific antisera and in the suppression of fertility (Goldberg et al., 1981; Goldberg, 1973). Replacement of the

* This work was supported by National Institutes of Health grants (to E. G . ) and a National Institutes of Health training grant predoc- toral fellowship (to H. H,). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t Present address: Dept. of Biochemistry, University of Iowa, Iowa City, IA 52241. I To whom correspondence should be addressed. ’ The abbreviations used are: LDH, lactate dehydrogenase; ELISA,

enzyme-linked immunosorbent assay; Boc, t-butoxycarbonyl; FMoc, fluoren-9-ylmethoxycarbonyl; HPLC, high performance liquid chromatography.

natural isozyme with a synthetic antigen would ensure avail- ability and homogeneity of vaccine preparations, as well as guarantee antigenic specificity for the testicular LDH isozyme (Goldberg et al., 1983).

Peptides, coupled to carrier molecules, have been shown to elicit the production of antibodies which will bind to the protein from which they were derived (Sutcliffe et al., 1983). This observation has raised considerable interest in develop- ing synthetic peptide vaccines (Lerner, 1984; Arnon, 1986). For example, immunization with peptides encompassing sequences of human chorionic gonadotropin (Stevens, 19861, influenza virus hemagglutinin (Shapira et al., 1984), foot-and- mouth disease virus VP1 (Bittle et aZ., 1982; DiMarchi et al., 1986), hepatitis B surface antigen (Gerin et al., 1983; Itoh et al., 1986), herpes simplex glycoprotein D (Eisenberg et al., 1985), and the malaria circumsporozoite protein (Egan et al., 1987) has been shown to protect experimental animals against subsequent challenge with the intact antigen.

The potential development of synthetic vaccines has fueled renewed interest in defining and predicting the antigenic sites of proteins, particularly those which can be represented by linear peptides (Berzofsky, 1985; Van Regenmortel, 1987). Immunological self-tolerance dictates that antigenic sites are located in regions of the molecular surface that differ struc- turally from the host’s self proteins (Benjamin et al., 1984). Crystallographic analyses of antigen-antibody complexes re- veal that an antigenic site may consist of as many .as 17-20 amino acids and can encompass as much as 750 A’ of the protein surface (Mariuzza et al., 1987; Sheriff et al., 1987; Colman et al., 1987; Amit et at., 1986).

A number of predictive approaches have been employed to identify potential antigenic determinants. Algorithms aimed at identifying continuous antigenic sites are based upon attempts to correlate peptide antigenicity with various structural features of protein antigens. For proteins whose structures are known at the amino acid level, some success in identifying surface epitopes has been achieved from prediction based on hydrophilicity (Hopp and Woods, 1981; Kyte and Doolittle, 1982) or the propensity to form a /3-turn (Gamier, et al., 1978).

In proteins of known three-dimensional structure, the accessibility of peptides to antibody-sized molecules has been shown to correlate with antigenicity. In these studifs, expo- sure was determined with a spherical probe of 10-A radius (Novotny et al., 1986) or through the use of protrusion indices (Thornton et al., 1986).

In addition to surface accessibility, a correlation has been observed between antigenicity and atomic mobility determined from the temperature factors of highly refined crystal structures (Westhof et al., 1984; Tainer et al., 1984). An extensive study employing myohemerythrin (Geysen et ai.,

10513

10514 Immunogenicity of LDH-G Sequences

1987), a molecule which is completely foreign to the vertebrate immune system, showed that those epitopes which are best mimicked by short peptides (6-10 residues) correspond to segments of the polypeptide chain which exhibit high flexibility, a convex surface shape, and negative electrostatic potential.

The parameters that dictate which peptides will be the most likely to elicit protein-reactive antibodies (immunogenic) are less clear. In this publication, we describe the immunological properties of a panel of synthetic peptides derived from mouse LDH-C4. These sequences were selected based upon the location of known antigenic sites and regions of high surface accessibility, segmental flexibility, and evolutionary variability. The relative immunogenicity of the peptides (as assessed by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, an9 Western blot) were compared with features of the refined 3-A structure of mouse LDH-C, (Hogrefe et al., 1987) to provide guidelines for selecting immunogenic LDH- Cq peptides for vaccine studies in humans and non-human model systems.

MATERIALS AND METHODS

All Boc-protected L-amino acids were purchased from Vega Bio- technologies, Inc. (Tucson, AZ) or Bachem (Torrance, CA). FMoc- amino acids were from Cambridge Research Biochemicals (Cam- bridge, Great Britain). p-Alkoxybenzyl alcohol polystyrene resins were purchased from Vega Biotechnologies, substituted with the appropriate Boc- or FMoc-amino acid (0.4-0.5 meq/g), respectively.

Solvents and chemicals were obtained from the following sources: Sequanal-grade trifluoroacetic acid and diisopropylethylamine (Chemical Dynamics Corporation); gold label indole, l-hydroxybenzotriazole, 1,3-dicyclohexylcarbodiimide (99%), pentafluorophenol (99+ %), and piperidine (98%) (Aldrich); HPLC/Spectro-grade dichloromethane, and dimethylformamide (Pierce Shemica1 Co.). An- hydrous dimethylformamide was stored over 4-A molecular sieves, bubbled daily with Nz, and tested prior to use with dinitrofluoroben- zene for the presence of secondary amines.

Peptide Synthesis-Seven peptides of mouse LDH-C4 were synthesized by solid-phase methodologies (listed in Fig. 1). Peptides 1, 3, 6, and 8 were assembled by the Boc-benzyl procedure (Barany and Merrifield, 1979; Stewart and Young, 1984). Peptides 2,7, and 9 were synthesized using the FMoc-t-butyl strategy (Chang and Meienhofer, 1978; Atherton et ai., 1983). All couplings were carried out with preformed Boc- or preformed FMoc-amino acids, which were activated as their symmetrical anhydrides, hydroxybenzotriazole esters, and/or pentafluorophenyl esters and added manually to the reaction vessel. Double couplings were routinely carried out using 5-6 equiv- alents of activated amino acid for each cycle. Completeness of the coupling reaction was monitored with the Kaiser ninhydrin test (Kaiser et al., 1970). The peptide resin was washed and filtered using either a Vega coupler model 1000 peptide synthesizer or a custom- made solvent delivery system. A cysteine residue was added to the amino terminus of each peptide to facilitate conjugation to a macromolecular carrier. In most cases, the S-ethylmercapto group, which remains intact during acid cleavages of peptides from the resin, was used for protection of the sulfhydryl moiety. Purification of crude cysteine-containing synthetic peptides are thus simplified as no dimerization and/or polymerization products contaminate the preparation. Before conjugation, the purified peptides are reduced in the presence of excess dithiothreitol, gel-filtered on Sephadex G-15 in 0.1 M acetic acid and lyophilized.

Boc-benzyl Synthesis-At each synthesis cycle, the a-amino group was deprotected with 50% trifluoroacetic acid/dichloromethane, containing 10% indole (Stewart and Young, 1984), and neutralized with 5% diisopropylethylamine/dichloromethane. Boc-amino acids were coupled as activated symmetric anhydrides (Hagenmaier and Frank, 1972) and 1-hydroxybenzotriazole esters (Konig and Geiger, 1970) for the first and second couplings, respectively. Final cleavage of the peptide from the resin and side chain deprotection was carried out with HBr/trifluoroacetic acid, containing 10% anisole (Stewart and Young, 1984).

FMoc t-Butyl-At each synthesis cycle, deprotection of the base- labile FMoc moiety was carried out with 20% piperidine/dimethylformamide. Activated FMoc-amino acids were coupled as symmetric

anhydrides (Chang and Meienhofer, 1978) and pentafluorophenol esters (Kisfaludy and Schon, 1983) for the first and second couplings, respectively. FMoc-glycine and FMoc-lysine were coupled as pentafluorophenol esters exclusively. All couplings were carried out for 1 h in dimethylformamide to minimize aggregation of the growing peptide chains (Pillai and Mutter, 1981). Final cleavage and side chain deprotection were carried out with 50% trifluoroacetic acid/dichloromethane, containing 10% anisole (Atherton et al., 1983).

Purification and Characterization of Synthetic Peptides-The crude cleavage produce, after rotary evaporation, was precipitated with cold anhydrous ethyl ether. The peptide mixture was extracted into acetic acid:water (3:1), washed several times with ether to remove anisole, and lyophilized.

The desired peptide was purified from modified and deletion peptides by reverse-phase HPLC. Crude peptides were first chromato- graphed on an analytical reverse-phase C,, column (Waters WBond- apak) in the presence of 0.1% trifluoroacetic acid (Pierce Chemical Co.) and eluted with a gradient of 0-50% acetonitrile.

Purification on a larger scale was accomplished using a semi- preparative CIS column (Waters gBondapak 19 X 150 mm) with typical loadings of 50-100 mg of crude peptide. HPLC runs were carried out at a flow rate of 10-20 ml/min based upon the optimal separation conditions of the analytical runs. Fractions were collected, pooled, and lyophilized.

With one exception, fractions used for immunochemical analysis were of greater than 95+ % purity, as judged by analytical reverse- phase HPLC and amino acid analysis. Chromatographic analysis of peptide 6 showed extensive dimerization and polymerization, and the desired sequence probably accounted for less than 50% of the total product.

HPLC-purified peptide samples (50-100 pmol) were hydrolyzed (110 "C, 24 h in uacuo) and derivatized with phenyl isothiocyanate (Pierce Chemical Co.). Phenyl isothiocyanate-amino acid derivatives were separated using the Waters PicoTag system (Cohen et al., 1984) and quantified relative to amino acid standards (Pierce Chemical Co.). The amino acid compositions of the HPLC-purified peptides were consistent with expected theoretical values.

Preparation of Antigens-In addition to the seven sequences described above, synthetic peptides 4 and 5 (Fig. l) were purchased from Peninsula Laboratories (San Carlos, CA) and judged to be homogeneous, as described previously (Wheat et aZ., 1985). A cysteine residue was subsequently added to the NHZ terminus of peptide 4 for conjugation purposes (Wheat et al., 1985). An unrelated peptide corresponding to residues 92-103 of tobacco hornworm moth cytochrome c (peptide 10, Fig. 1) served as a control sequence.

Peptides 2, 3, 4, 5, 7, and 8 were conjugated to a macromolecular carrier, using a previously described method (Wheat et al., 1985). Briefly, the reduced purified peptides were coupled to diphtheria toxoid (Connaught Laboratories, Philadelphia, PA) through their amino-terminal sulfhydryl groups with the heterobifunctional reagent 6-maleimidocaproyl N-hydroxysuccinimide (Sigma) (Lee et ai., 1980). The degree of substitution was measured using Ellman's reagent, as described (Gonzales-Prevatt et al., 1982) as well as amino acid analysis. Coupling efficiencies ranged from 12 to 20 peptides/carrier molecule.

LDH-C4 was purified to crystalline homogenity from random-bred mouse testes by affinity chromatography on 5'-AMP-Sepharose (Bachman and Lee, 1976).

Preparation of Antisera-Six groups of female rabbits (Langshaw Farms, Augusta, MI), consisting of between one to three animals each, received primary immunizations at multiple intradermal sites of 2 mg of peptide-diphtheria toxin conjugate emulsified in complete Freund's adjuvant. Secondary immunizations consisted of 1 mg of conjugate in incomplete Freund's adjuvant and were administered in the same manner 4 weeks after the primaries. Sera were collected at regular intervals, heated at 56 "C for 30 min to destroy complement, and aliquots stored at -20 "C until assayed. Two +21 day sera were pooled from rabbits receiving identical formulations and tested for: (a ) reactivity with the immunizing peptide and mouse LDH-Ca by an ELISA, (b) reactivity with mouse LDH-C4 by immunoprecipitation of '251-labeled LDH-C,, and ( c ) specificity for the mouse LDH-C, protein by Western blotting of mouse tissue extracts.

Rabbit antisera to affinity purified mouse LDH-C4 were prepared as described (Liang et al., 1986). The ELISA procedure was adapted from Liang et al. (1986). Results were expressed as the antiserum dilution which gave an arbitrary ELISA reading on a Dynatech ELISA reader.

A solution-phase radioimmunoassay was employed with polyeth-

Immunogenicity of LDH-C4 Sequences 10515

ylene glycol ( M , 6000-7500) as the precipitating agent and ‘251-labeled mouse LDH-C4 as tracer (Liang et al., 1986; Wheat et al., 1985). All samples were assayed in triplicate. Peptide-specific antibody binding to LDH-C4 was expressed as the total counts/min precipitated by the anti-peptide antiserum minus the counts/min precipitated in the presence of an excess amount of the homologous peptide. Results were also calculated as the mean micrograms of ‘251-labeled LDH-Cd bound per ml of serum.

Western Blot Analysis-The LDH enzyme activity of crude mouse testes and liver homogenates was measured spectrophotometrically as described (Hawtrey and Goldberg, 1970). Extracts (0.5 units of enzyme activity/Iane) were electrophoresed under native conditions using 7.5% polyacrylamide gel electrophoresis gels. Protein blotting was done according to the method of Burnette (1981) except that electrophoretic transfer to nitrocellulose (Schleicher and Schuell) was performed with cooling in native running buffer (5 mM Tris, 40 mM glycine, pH 8.3).

One nitrocellulose strip was stained for LDH activity (Goldberg, 1964). Total protein was detected by Amido Black staining.

The remaining strips were blocked with 5% bovine serum albumin, rinsed with Tris-buffered saline, incubated with peroxidase conjugated to goat anti-rabbit IgG (Boehringer Mannheim), and then in a substrate solution prepared with 4-chloro-1-naphthol and hydrogen peroxide as described (Monroe, 1985).

RESULTS

The three-dimensional structure of mouse LDH-C4 has been determined (Musick and Rossmann, 1979; Hogrefe et al., 1987). Temperature factor and surface accessibility pro- files for the refined 3-A structure of mouse LDH-C4 have been published (Hogrefe et al., 1987). Previous attempts to correlate features of the structure with the location of antigenic sites were based upon the binding of anti-mouse LDH-C4 antibodies to tryptic peptides by solid-phase radioimmunoassay (Wheat et al., 1985). To obtain a more complete map of the antigenic determinants on LDH-C4, a panel of synthetic peptides was selected for synthesis based on a number of criteria which have been shown to correlate with antigenicity.

Structural analysis of mouse LDH-C4 showed that, in general, amino acids which are accessible to solvent in th? native tetramer (determined with a spherical probe of 1.4-A radius to simulate a water molecule; (Lee and Richards, 1971)) exhibit a high degree of flexibility, whereas those residues located in the protein interior or in the subunit interfaces have lower temperature factors. For LDH-C4, nine major peaks of B value maxima are centered at positions 8-20, 51- 55, 79-82, 96-99, 115-124, 150-151, 207-228, 282, and 315- 330 (Hogrefe et al., 1987). In addition, regions of the tetramer which are accessible to antibody-sized yolecules have been identified by calculation relative to a 10-A radius probe (No- votny et d., 1986) and correspond to the following sequences: 1-16, 51-57, 79-82, 97-117, 124, 150, 214-241, 276-284, and 308-330 (Hogrefe et al., 1987).

Nine peptides encompassing the most flexible and accessible segments of the LDH-C4 tetramer, and hence the most likely to be immunogenic, were synthesized (listed in Fig. 1). Each of these peptides encompasses between 10 and 15 amino acids of mouse LDH-C4 and, in total, represent >50% of the solvent accessible surface of the tetramer. In addition, these synthetic peptides comprise sequences, which span the amino and carboxyl termini, exhibit a variety of secondary structural attributes and cover virtually all the prominent maxima of the hydrophilicity profile (Hopp and Woods, 1981; Kyte and Doolittle, 1982). Sequence variability in the synthetic peptides ranged from 40 to 60% and was calculated as the average number of variations in each peptide in pairwise comparisons with somatic LDH sequences from human, murine, and por- cine sources. Although the complete amino acid sequences of rabbit (the host’s) somatic LDHs are not known, the infor- mation which is available (Taylor and Oxley, 1976; Brummel

- Eplircpn - a b

1 C(SEt)-l-l4b c(SEt)-S-T-V-K-E-Q-L-I-Q-N-L-V-P-E-D

2 C(SEt)-5-15

3 C(SEe)-C-49-58 C(SEt)-C-V-D-A-D-T-D-K-L-8-C

4 97-110 R-H-V-S-C-Q-T-B-L-D-L-L-9-R

5 c-211-220 C-S-L-N-P-A-I-C-T-D-Y

a b C(SEt)-E-Q-L-I-Q-A-L-V-P-E-D-K

6 C-231-243 C-9-V-V-E-C-C-Y-E-V-L-D-H-K

7 C(SEt)-C3-276-286-C C(SXt)-C-C-C-L-V-K-G-F-H-C-I-K-E-E~V-F-C

8 C(SEt)-3D4-316 C(SEt)-V-N-H-T-A-E-E-E-C-L-L-K-K

9 C(SEt)-318-330 C(SEt)-A-D-T-L-U-N-H-Q~~-N-L-E-L

10 C(SEt)-cc92-103 C(SEC)-A-D-L-I-A-Y-L~K~Q~A~~-K

FIG. 1. Amino acid sequences of the assembled mouse LDH- C4 peptides. In the text, the synthetic peptides are referred to as numbers 1 to 9, as indicated. Control peptide 10 corresponds to sequence 92-103 of tobacco horn worm moth cytochrome c (cc). C(SEt) refers to an S-ethylmercapto-protected NH,-terminal cysteine residues. The positions of glycine spacers (C) and cysteine linkers (C), not present in the natural mouse LDH-C4 sequence, are indicated.

+ ... . . . . . . . . . . . . . . . +

+ I f 4 / ........................................................................................

e + 2 * ....... * . . . . . . ...................................................... ..* .......................... q;y;*$L;;;*l

0 1 2 3 4 6 8 7 8 8 1 0

LDX-C‘ PEPTIDES

FIG. 2. Antigenicity of mouse LDH-C4 synthetic peptides. The amino acid sequences of the peptides are given in Fig. 1. The binding of rabbit anti-mouse LDH-C4 antibodies was measured by ELISA. The results are expressed as the antiserum dilution which gave an absorbance of 0.2 units above background (corrected by subtracting out the absorbance of preimmune sera at the same dilution). The three different serum pools tested exhibited the following LDH-C4-specific titers: 0, >> 21,870 (pool from two rabbits); +, 240,000 (pool from two rabbits); *, 180,000 (pool from four rabbits).

and Stegnink, 1970) shows that rabbit LDH-A4 is indeed homologous to the known vertebrate LDH-AI molecules.

The relative antigenicity of the synthetic peptides (capacity to bind antibodies raised against the intact protein) was determined by ELISA (Fig. 2). The binding by rabbit anti- mouse LDH-C4 antibodies to the synthetic peptides can be evaluated both in terms of the antibody titer and the number of antisera which show cross-reactivity (Geysen et al., 1987). By using these two criteria, the synthetic peptides can be grouped according to their ability to bind rabbit anti-mouse LDH-C4 antibodies: 1) “highly antigenic” (react with 3/3 sera pools; mean antibody titer in the range of 1425-3300), peptides 1, 2, 6, and 8; 2) “moderately antigenic” (react with 1- 3/3 sera pools; mean antibody titer in the range of 640-1670), peptides 3, 5, 7, and 9; 3) “nonantigenic” (no reactivity observed), peptides 4 and 10 (control sequence).

The binding of rabbit anti-mouse LDH-C4 antibodies to the synthetic peptides was also evaluated in a solution-phase competition assay using lZ5I-labeled mouse LDH-C4 as a tracer. This assay allows an estimate of the relative affinities

10516 Immunogenicity of LDH-C4 Sequences

of antibodies for the peptide versus the native protein. None of the synthetic peptides (at concentrations ranging from 10"' to lo-, M ) were able to displace the binding of rabbit anti-mouse LDH-C, antibodies from Iz5I-LDH-C4. No competition was observed even when the nine peptides were pooled together, using concentrations which were 104-fold higher than the amount of unlabeled LDH-C4 required to completely saturate all available antibody combining sites. Taken together these results indicate that antibodies raised to the native LDH-C, bind to peptides when assayed by ELISA but have no or low affinity for the peptides in solution. A reasonable conclusion is that these short peptides do not faithfully mimic the conformation of the corresponding epitope in the native protein. Alternatively, the antibodies may be detecting denatured antigen by solid-phase assay (as antigen attaches to the plate) or from the immunizing preparation (when antigen is emulsified in Freund's adjuvant). Indeed a recent study has questioned whether a protein antigen is in its native state when assayed for antibody binding (Jemmer- son, 1987). The discrepancy between the ELISA and solution- phase assays emphasizes the need to use a variety of immunochemical techniques when the antigenicity of peptides and proteins is measured (Van Regenmortel, 1987).

The relative immunogenicity of six mouse LDH-C4 sequences was determined in rabbits (Table I). All of the rabbits received diphtheria toxin-peptide conjugates that had been prepared and administered in an identical fashion. Examina- tion of individual responses (Hogrefe, 1987; Wheat and Gold- berg, 1984; Wheat et al., 1985) showed that although there was variability among animals receiving the same formula- tion, in all cases the magnitude of the immune response appeared to differ between animals receiving different peptide conjugates. To compensate for variability in the immune response and enable general trends to be examined, antiserum was pooled from two or three rabbits (only one rabbit was immunized with peptide 3). Similar investigations aimed a t comparing the immune response to different peptides (Wes- thof et al., 1984) have used serum pools from the same number of animals.

Antibody titers against the appropriate peptide immunogen and against mouse LDH-C, were measured by ELISA (Table I). All of the antisera contain antibodies which bind to the immunizing peptide and the intact LDH-C, molecule. Levels of LDH-C4-specific antibody appear to be highest in the sera of rabbits immunized with peptides 2 and 8 and lowest in

TABLE I Immunogenicity of selected mouse LDH-C4 peptides in rabbits

'"I-LDH-CI ?.l̂ titeP immunoprecipitation

Antiserum '*"' . .

pooled Peptide LDH",, Peptide- '*'I-LDH-C, specificb bound

LDH-C, Total pools Pools

2 8 5 7 4 3

2 15

3 3 3 2 3 1

<50 700

1,780 1,400 1,900

960 700 980

17,000 720

2,100 2,200 1,250

560 335 28

precrprtated Cpll

ND 5,393

3,227 3,301 2,258 1,259

0 0

pglml serum

378 0.048

0.029 0.029 0.020 0.011 0 0

ELISA titer is expressed as the serum dilution which gives 50% maximal binding.

* Calculated as (counts/min precipitated by 40 p1 of anti-peptide antiserum) (counts/min precipitated in the presence of excess peptide

M)).

rabbits immunized with peptides 3 and 4. An identical hierarchy of reactivity with LDH-C4 was also observed with anti- peptide antisera collected after tertiary immunizations.

Antibody titers were also determined by immunoprecipitation of '2sI-labeled LDH-C4 (Table I). Antisera from rabbits immunized with peptides 2,5,7, and 8 specifically precipitated '2sI-labeled LDH-C,, whereas antibodies to peptides 3 and 4 did not. Antibody binding to LDH-C4 could be absorbed with the homologous peptide, in a concentration-dependent manner (only the data with excess amount of competing peptide are shown in Table I), thereby demonstrating that the anti- peptide antibodies can bind in solution to both the peptide immunogen and the corresponding epitope in the intact molecule. The relative affinities of the different antisera for LDH- C4 in the solution-phase assay were similar to those seen with the ELISA (Table I) and consistent with the hierarchy of immunogenicity established previously for peptides 2, 4, and 5 (Wheat et al., 1985).

Antibodies raised against LDH-C, and the peptide conjugates bind to LDH-C, in solution with affinities which differ by a t least four logs (Table I). A pool of the six different site- specific sera could precipitate more of the protein than any of the sera individually, although the binding was still a fraction of that obtained with antibodies raised against the whole protein.

The specificity of the anti-peptide antisera for mouse LDH- C4 was also evaluated with Western blotting techniques (Fig. 3). Mouse testes and somatic tissue extracts were electrophoresed under native conditions to separate the various LDH isozymes and transferred to nitrocellulose. LDH activity staining of the strips showed that the testes extract lanes contained only the C4 isozyme, whereas the liver extract lanes contained the A, isozyme (migrates close to mouse LDH-C4) and minor amounts of A/B heterotetramers. The strips were incubated with dilutions of anti-LDH-C4 and anti-peptide antisera which bind mouse LDH-C4 similarly in the ELISA (Table I). Fig. 3 shows that each of the anti-peptide antisera binds to mouse LDH-C4. With the exception of antisera raised to peptide 3, which has the lowest ELISA titer for mouse LDH-C, (Table I), binding to somatic LDHs or other unrelated proteins was not observed a t these dilutions, thereby

1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

FIG. 3. Western blotting of mouse tissue extracts with rabbit anti-mouse peptide antisera. Mouse testes or liver extracts were electrophoresed in lunes T and L, respectively. Strip I was stained for LDH activity. The remaining strips were incubated with sera from rabbits immunized with the following: 2, mouse LDH-C4 (diluted 1/7000); 3, peptide 8 (1000); 4, peptide 2 (1000); 5, peptide 5 (500); 6, peptide 6 (500); 7, peptide 4 (100); 8, peptide 3 (100). Strip 9 was incubated with preimmune rabbit sera (diluted 1/100).

Immunogenicity of LDH-CI Sequences 10517

demonstrating the specificity of the anti-peptide antisera for the mouse testicular isozyme.

Different molecular properties were examined for their correlation with the immunological activities of the panel of synthetic peptides (Table 11).

Peptides 2 and 8 were the most antigenic sequences (Fig. 1) and elicited the highest LDH-C4-specific antibody titers in rabbits when used as immunogens (Table I). The molecular feature which appears to distinguish these peptides, from the moderately active group consisting of peptides 5 and 7, is the degree of sequence variability from the corresponding region in somatic LDHs. However, no satisfactory explanation for the poor reactivity of peptide 4 was found based upon its intrinsic structural properties or its evolutionary divergence from the somatic isozymes.

DISCUSSION

The relative immunogenicity of a peptide has been attrib- uted both to the intrinsic properties of an antigen and to extrinsic factors characteristic of the host (Berzofsky, 1985). The role of inherent structural features and sequence variability in determing the immune response to mouse LDH-C4 and its peptide homologs was examined in order to identify sequences likely to be useful as synthetic vaccines. Attempted correlations were made with the immunological reactivity of two different sets of peptides. Previous correlations were based upon a panel of randomly generated tryptic fragments, which varied in length and physical properties (Hogrefe et al., 1987). The molecular feature that appeared to distinguish the most antigenic fragments from the least was mean a5cessible surface area in the monomer, measured with a 10-A radius probe. The presence of epitopes on the tetramer surface and at the subunit interfaces indicated that LDH-C, is recognized by the immune system as a partially dissociated species, presumably the result of tetramer dissociation in Freund's adjuvant (Hogrefe et al., 1987).

The results presented here describe the immunological properties of a panel of synthetic peptides that correspond to the most accessible and flexible segments of the LDH-C4 molecule. Only these peptides which are exposed in the tetramer were chosen for study, since antibody formation was measured to the physiologically relevant form. As expected and unlike the outcome with peptides from the completely foreign myohemerythrin (Geysen et al., 1987), immunological reactivity appeared to be determined by immunological self- tolerance. Peptides 2 and 8 (residues 5-15 and 304-316, respectively), which occupy portions of the polypeptide chain that have diverged evolutionarily to the greatest extent (Ev- entoff et al., 1977), were found to be the most antigenic and immunogenic. Peptides which were less reactive corresponded to regions of LDH-C4 that exhibit fewer amino acid differences from the somatic forms.

The influence of immunological self-tolerance on the reactivity of mouse LDH-C, peptides is consistent with results obtained by other investigators with intact antigens, like cytochrome c (Jemmerson and Margoliash, 1979), myoglobin (Cooper et al., 1984), and lysozyme (White et al., 1978). These studies have shown that the immune response to evolutionarily conserved antigens is biased towards regions of the molecule which exhibit structural differences from the host's self proteins. In the studies described here with mouse LDH- C4 peptides, immunological self-tolerance appeared to dictate the probability that antibodies were raised to a given site when the whole protein was used for immunization and that protein-specific antibodies were elicited when the peptide sequence was used as an immunogen. The influence of immunological self-tolerance on the immunological activity of moust LDH-C, peptides precluded an assessment of the relative roles of surface accessibility and segmental flexibility.

Immunological self-tolerance and intrinsic structural features, however, could not account for the poor reactivity in rabbits of peptide 4 (residues 97-110), which is highly acces-

TABLE I1 Immunological and structural properties of moue LDH-C4 DeDtides

Peptide Surface accessibility'

No." Average

factord Relative immu- Len,& Secondary Total, Average temperature Sequence variability'

nogenicityb structure 1.4A 1.4A IOA A' A' %

Highly antigenic 1 ND 15 la, extended 945 8.4 8.6 27.3 2 1 12 Extended 868 9.0 6 ND 13 la

9.7 31.1 536 5.1 2.3

8 1 13 la 703 7.3 9.1 16.7 13.9

Moderately antigenic 3 4 10 Loop 554 6.7 5.7 18.3 5 2 10 Irregular 293 5.7 2.0 24.1 7 3 13 Loop 533 5.3

4 4 14 Loop 862 8.6 8.6 19.0

9 ND 7.2 14.9

1,002 9.1 14.2 33.1 13 la Nonantigenic

LDH-C, 331 NA 11,146 3.6 4.4 15.7 Peptide numbers correspond to those given in Fig. 1.

* Relative immunogenicity was ranked (1-4) in order of decreasing immunogenicity (1 > 2, etc.), as determined in Table I; ND, not determined; NA, not applicable.

Surface accessibility was calculated for the mouse LDH-Ca tetramer relative to probes of 1.4- and 10-A radii (Hogrefe et al., 1987). Average accessibility per atom is the total accessibility of the peptide divided by the number of non-hydrogen atoms.

dAverage temperature factor per main chain atom in the peptide (Hogrefe et al., 1987). e The average number of variant sites in mouse LDH-C, in painvise comparisons with sequences of pig (Eventoff

et al., 1977), human (Tsujibo et al., 1985, Sakai et al., 1987), and mouse (Akai et al., 1985, Fukasawa and Li, 1986; Li et al., 1985) LDH-AI and LDH-B4. Results are expressed as a percentage of the total number of residues in the peptide.

57 63 46 58

40 44 41 40

50 30

10518 Immunogenicity of LDH-C4 Sequences

53 * * 58

11. HC A--L--D--K--L--K HA l K - - - - HI L E - - - -

309 * * * * 314 319 314 * * 328 331. * * * * VII. HC S--~--L--C--A--L-.F--K--K.-S--A~-E--T~.L.-U--N-~I~~Q--K~.D--L.-I-~F

HA - . - - - R L - - - . D . . - . C . - - K - Q - D D . V - Q L - - - . D - - - D . - - - - K D

FIG. 4. Amino acid sequence variability in the putative epitopes of human LDH-Cr. The seven most accessible segments of the mouse LDH-C, molecule (Hogrefe et al., 1987) are shown, with the corresponding amino acid sequences of human LDH-C4 (HC) (Millan et al., 1987), human LDH-A4 ( H A ) (Tsujibo et al., 1985), and human LDH-Ba ( H B ) (Sakai et al., 1987). Asterisks mark the positions of residues which are particularly accessible to a 10-8, radius probe in mouse LDH-C,.

sible, flexible, and exhibits several differences in amino acid sequence from the corresponding region in vertebrate somatic LDHs. However, both polyclonal (data not shown) and mono- clonal (Goldman-Leikin and Goldberg, 1983) antibodies raised in mice against mouse LDH-C4 bind to this synthetic peptide. Thus, other host regulatory mechanisms (reviewed in Benjamin et al., 1984) appear to be influencing the immune response in rabbits to peptide 4.

Once the immunological properties of LDH-C, and its peptide homologs had been defined in an experimental animal model system, it was of interest to extrapolate these results to contraceptive vaccine development in the human. Since antibodies raised to mouse LDH-C4 peptides show a dramatic decrease in binding to rat LDH-C4 (Hogrefe, 1987), it seems reasonable to assume that peptide immunogens which are homologous to human LDH-C, will be the most effective as vaccines designed for human use. The high degree of sequence homology between mouse and human LDH-C4 (74%) (Millan et al., 1987) and conservation of structure among the LDH isozymes (Musick and Rossmann, 1979) suggests that the molecular features of the refined mouse LDH-C4 structure should be nearly identical to those of the human testicular isozyme. Thus, the importance of intrinsic factors and immunological self-tolerance, which determined the outcome of the immune response in rabbits to mouse LDH-C4 peptides, may be applied to vaccine development with human LDH-C4.

Fig. 4 shows the segments of the LDH-C4 structure, which are particularly accessible to antibody-sized molecules in the tetramer and the corresponding amino acid sequences of human LDH-C4, LDH-A4, and LDH-B4. Comparative se-

quence analysis among the LDHs (Millan et al., 1987) shows that human LDH-C4 and LDH-A, are more closely related (75% homology) than mouse LDH-C4 is to the seven somatic LDHs of known sequence (59-72% homology) (Li et a/., 1983). Only domain I, which encompasses residues 1-16, exhibits a considerable amount of sequence variability from the corresponding regions of human somatic isozymes. Therefore, this peptide appears to be the most obvious candidate for a linear synthetic peptide vaccine against LDH-C4, designed for human use.

Although peptides encompassing the flexible, accessible, and evolutionarily variable segments of LDH-C4 elicit protein-reactive antibodies, they appear to be much less immunogenic than the intact protein (Table I). This is presumably due to the structural disparity between small, unstructured peptides and complex protein epitopes (Jemmerson and Pat- terson, 1986), which have been recently determined by crystallographic analyses (Amit et al., 1986; Mariuzza et al., 1987; Sheriff et al., 1987; Colman et al., 1987). Peptides which more closely mimic the conformation of surface regions may elicit antibodies which bind with higher affinity to the intact protein. To address this question, research is currently focused on comparing the immunogenicity of short, unstructured peptides and sequences designed to mimic native secondary and tertiary

Acknowledgments-We are most grateful to Thomas E. Wheat for his enthusiastic encouragement and to Michael Rossmann for making his laboratory available to Holly Hogrefe for the molecular graphics. We wish to thank Martine Benoit for preparation of this manuscript.

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