Crystal structures of pheasant and guinea fowl egg-white lysozymes.

The crystal structures of pheasant and guinea fowl lysozymes have been determined by X-ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6(1)22 with cell dimensions a = 89.2 A and c = 61.7 A. The structure was refined to a final crystallographic R-factor of 17.0% for 8,854 observed reflections in the resolution range 6-1.9 A. Crystals of pheasant lysozyme are tetragonal, space group P4(3)2(1)2, with a = 98.9 A, c = 69.3 A and 2 molecules in the asymmetric unit. The final R-factor is 17.8% to 2.1 A resolution. The RMS deviation from ideality is 0.010 A for bond lengths and 2.5 degrees for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N-terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3-dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the C alpha atom of Gly 41 and absent in lysozymes from other species (which have a large side-chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other c-type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody-bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen-antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation in c-type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes.     

The primary structure of a novel goose-type lysozyme from rhea egg white

G-type lysozyme is a hydrolytic enzyme sharing a similar tertiary structure with plant chitinase. To discover the relation of function and structure, we analyzed the primary structure of new G-type lysozyme. The complete 185 amino acid residues of lysozyme from rhea egg white were sequenced using the peptides hydrolyzed by trypsin, V8 protease, and cyanogen bromide. Rhea lysozyme had sequence similarity to ostrich, cassowary, goose, and black swan, with 93%, 90%, 83%, and 82%, respectively. The six substituted positions were newly found at positions 3 (Asn), 9 (Ser), 43 (Arg), 114 (Ile), 127 (Met), and 129 (Arg) when compared with ostrich, cassowary, goose, and black swan lysozymes. The amino acid substitutions of rhea lysozyme at subsite B were the same as ostrich and cassowary lysozymes (Ser122 and Met123). This study was also constructed in a phylogenetic tree of G-type lysozyme that can be classified into at least three groups of this enzyme, namely, group 1; rhea, ostrich, and cassowary, group 2; goose, black swan, and chicken, and group 3; Japanese flounder. The amino acid sequences in assembled three alpha-helices found in this enzyme group (Thammasirirak, S., Torikata, T., Takami, K., Murata, K., and Araki, T., Biosci. Biotechnol. Biochem., 66, 147-156 (2002)) were also highly conserved, so that they were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.     

An antigenic determinant common to human chorionic somatomammotropin and human growth hormone revealed by limited proteolysis of immune complexes.

An epitope of human chorionic somatomammotropin for one of the monoclonal antibodies raised against the whole antigen has been identified. We compared the release of peptides from limited proteolysis of the antigen in the presence and absence of the related antibody. Using enzymes of different specificity, we could determine the amino acid sequence that can be considered at least inclusive of the epitope. The monoclonal antibody selected is 100% cross-reactive with human growth hormone, so the antigenic determinant identified is shared by the two protein hormones.     

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and its complexes with various species-variant and mutant lysozymes have been the subject of considerable experimental and theoretical investigation. The affinity of HyHEL-5 for bobwhite quail lysozyme (BWQL) is over 1000-fold lower than its affinity for the original antigen, hen egg lysozyme (HEL). This difference is believed to arise almost entirely from the replacement in BWQL of the structural and energetic epitope residue Arg68 by lysine. In this study, the association and dissociation kinetics of BWQL with HyHEL-5 were investigated under a variety of conditions and compared with previous results for HEL. HyHEL-5-BWQL association follows a bimolecular mechanism and the dissociation of the antibody-antigen complex is a first-order process. Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0 to 10.0) produced about a 2-fold change in the association and dissociation rates. The effect of viscosity modifiers on the association reaction was also studied. The large difference in the HEL and BWQL affinities for HyHEL-5 is essentially due to differences in the dissociation rate constant.     

Amino acid sequence of California quail lysozyme. Effect of evolutionary substitutions on the antigenic structure of lysozyme.

To examine the effect of amino acid substitutions in lysozyme on the binding of antibodies to lysozyme, we purified lysozyme from the egg whites of California quail and Gambel quail. Tryptic peptides were isolated from digests of the reduced and carboxymethylated lysozymes and subjected to quantitative analysis of their amino acid compositions. The two proteins were identical by this criterion. Each peptide from the California quail lysozyme was then sequenced by quantitative Edman degradation, and the peptides were ordered by homology with other bird lysozymes. California quail lysozyme is most similar in amino acid sequence to bobwhite quail lysozyme, from which it differs by two substitutions: arginine for lysine at position 68 and histidine for glutamine at position 121. California and bobwhite quail lysozymes were antigenically distinct from each other in quantitative microcomplement fixation tests, indicating that substitutions at one or both of these positions can alter the antigenic structure of lysozyme. Yet neither of these positions is among those claimed to account for the precise and entire antigenic structure of lysozyme [Atassi, M. Z., & Lee, C.-L. (1978) Biochem. J. 171, 429--434]. Two possible explanations for this discrepancy are discussed.     

On the attribution of binding energy in antigen-antibody complexes McPC 603, D1.3, and HyHEL-5

Using X-ray coordinates of antigen-antibody complexes McPC 603, D1.3, and HyHEL-5, we made semiquantitative estimates of Gibbs free energy changes (delta G) accompanying noncovalent complex formation of the McPC 603 Fv fragment with phosphocholine and the D1.3 or HyHEL-5 Fv fragments with hen egg white lysozyme. Our empirical delta G function, which implicitly incorporates solvent effects, has the following components: hydrophobic force, solvent-modified electrostatics, changes in side-chain conformational entropy, translational/overall rotational entropy changes, and the dilutional (cratic) entropy term. The calculated delta G ranges matched the experimentally determined delta G of McPC 603 and D1.3 complexes and overestimated it (i.e., gave a more negative value) in the case of HyHEL-5. Relative delta G contributions of selected antibody residues, calculated for HyHEL-5 complexes, agreed with those determined independently in site-directed mutagenesis experiments. Analysis of delta G attribution in all three complexes indicated that only a small number of amino acids probably contribute actively to binding energetics. These form a subset of the total antigen-antibody contact surface. In the antibodies, the bottom part of the antigen binding cavity dominated the energetics of binding whereas in lysozyme, the energetically most important residues defined small (2.5-3 nm2) "energetic" epitopes. Thus, a concept of protein antigenicity emerges that involves the active, attractive contributions mediated by the energetic antigenic epitopes and the passive surface complementarity contributed by the surrounding contact area. The D1.3 energetic epitope of lysozyme involved Gly 22, Gly 117, and Gln 121; the HyHEL-5 epitope consisted of Arg 45 and Arg 68. These are also the essential antigenic residues determined experimentally. The above positions belong to the most protruding parts of the lysozyme surface, and their backbones are not exceptionally flexible. Least-squares analysis of six different antibody binding regions indicated that the geometry of the VH-VL interface beta-barrel is well conserved, giving no indication of significant changes in domain-domain contacts upon complex formation.     

Amino acid sequence and immunological properties of chachalaca egg white lysozyme

Summary The amino acid sequence of lysozyme c from chachalaca egg white was determined. Like other bird lysozymes c, that of the chachalaca has 129 amino acid residues. It differs from other avian lysozymes c by 27 to 31 amino acid substitutions as well as by being devoid of phenylalanine. It contains substitutions at 9 positions which are invariant in the other 7 bird lysozymes of known sequence. Although the chachalaca is classified zoologically in the order Galliformes, which includes chickens and other pheasant-like birds, its lysozyme differs more from those of pheasant-like birds than do the lysozymes c of ducks. Phylogenetic analysis of the sequence comparisons confirms that the lineage leading to chachalaca lysozyme c separated from that leading to other galliform lysozymes c before the duck lysozyme c lineage did. This indicates a contrast between protein evolution and evolution at the organismal level. Immunological comparison of chachalacalysozyme c with other lysozymes of known sequence provides further support for the proposal that immunological cross-reactivity is strongly dependent on degree of sequence resemblance among bird lysozymes.103rd communication on lysozymes from the Laboratory of P. Jollès. Supported in part by grants from C.N.R.S. (ER 102), I.N.S.E.R.M. (Groupe de recherche U-116), N.S.F. (GB-42028X), and N.I.H. (GM-21509).     

Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia

The amino acid and cDNA sequences of lysozyme from the giant silk moth Hyalophora cecropia have been determined. This enzyme is one of several immune proteins produced by the diapausing pupae after injection of bacteria. Cecropia lysozyme is composed of 120 amino acids, has a mol. wt. of 13.8 kd and shows great similarity with vertebrate lysozymes of the chicken type. The amino acid residues responsible for the catalytic activity and for the binding of substrate are essentially conserved. Three allelic variants of the Cecropia enzyme are identified. A comparison of the chicken and the Cecropia lysozymes shows that there is a 40% identity at both the amino acid and the nucleotide level. Some evolutionary aspects of the sequence data are discussed.     

Effective reduction of antigenicity of hen egg lysozyme by site-specific glycosylation

Various mutant lysozymes were constructed by genetic modification and secreted in yeast expression system to evaluate the changes in the antigenicity of hen egg lysozyme (HEL). Although Arg68, the most critical residue to antigenicity of HEL, was substituted with Gln, the binding of monoclonal antibodies (mAbs) with the mutant lysozyme did not critically reduce, remaining 60% of the binding with mAb. In contrast, glycosylated mutant lysozyme G49N whose glycine was substituted with asparagine dramatically reduced the binding with mAb. The oligomannosyl type of G49N lysozyme reduced binding with mAb to one-fifth, while the polymannosyl type of G49N lysozyme completely diminished the binding with mAb. This suggests that the site-specific glycosylation of lysozyme in the interfacial region of lysozyme-antibody complex is more effective to reduce the antigenicity than the mutation of single amino acid substitution in the interfacial region.     

The amino acid sequence of wood duck lysozyme

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme.     

The Amino Acid Sequence of Wood Duck Lysozyme

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme.     

New variant of quail egg white lysozyme identified by peptide mapping

Two lysozymes were purified from quail egg white by cation exchange column chromatography and analyzed for amino acid sequence. The enzymes showed the same pH optimum profile for lytic activity with broad pH optima (pH 5.0-8.0) but had difference in mobility on native-PAGE. The native-PAGE immunoblot showed one or two lysozymes present in individual egg whites. The established amino acid sequence of quail egg white lysozyme A (QEWL A) was the same as quail lysozyme reported by Kaneda et al. [Kaneda, M., Kato, I., Tominaga, N., Titani, K., Narita, K., 1969. The amino acid sequence of quail lysozyme. J. Biochem. (Tokyo). 66, 747-749] and had six amino acid substitutions at position 3 (Phe to Tyr), 19 (Asn to Lys), 21 (Arg to Gln), 102 (Gly to Val) 103 (Asn to His) and 121 (Gln to Asn) compared to hen egg white lysozyme. QEWL A and QEWL B showed one substitution, at the position 21, Gln replaced by Lys, plus an insertion of Leu between position 20 and 21, being the first report that QEWL B had 130 amino acids. The amino acid differences between two lysozymes did not seem to affect antigenic determinants detected by polyclonal anti-hen egg white lysozyme, but caused them to separate well from each other by ion exchange chromatography.     

Stomach lysozymes of ruminants. II. Amino acid sequence of cow lysozyme 2 and immunological comparisons with other lysozymes

The complete sequence of 129 amino acids has been determined for one of three closely related lysozymes c purified from cow stomach mucosa. The sequence differs from those known for 17 other lysozymes c at 39-60 positions, at one of which there has been a deletion of 1 amino acid. The glutamate replacement at position 101 and the deletion of proline at position 102 eliminate the aspartyl-prolyl bond that is present between these positions in all other mammalian lysozymes c tested. This bond appears to be the most acid-sensitive one in such lysozymes at physiological temperature. Of the 40 positions previously found to be invariant among lysozymes c, only one has undergone substitution in the cow lineage. This modest number of changes at novel positions is consistent with the inference, based on tree analysis and antigenic comparisons, that the tempo of evolutionary change in the cow lysozyme lineage has not been radically different from that in other lysozyme c lineages. The mutations responsible for the distinctive catalytic properties and stability of cow lysozyme c could be a minor fraction of the total that have been fixed in the cow lineage.     

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.     

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.     

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.     

Crystallographic refinement of the three-dimensional structure of the FabD1.3-lysozyme complex at 2.5-A resolution.

The three-dimensional crystal structure of the complex between the Fab from the monoclonal anti-lysozyme antibody D1.3 and the antigen, hen egg white lysozyme, has been refined by crystallographic techniques using x-ray intensity data to 2.5-A resolution. The antibody contacts the antigen with residues from all its complementarity determining regions. Antigen residues 18-27 and 117-125 form a discontinuous antigenic determinant making hydrogen bonds and van der Waals interactions with the antibody. Water molecules at or near the antigen-antibody interface mediate some contacts between antigen and antibody. The fine specificity of antibody D1.3, which does not bind (K alpha less than 10(5) M-1) avian lysozymes where Gln121 in the amino acid sequence is occupied by His, can be explained on the basis of the refined model.     

Three distinct epitopes within the loop region of hen egg lysozyme defined with monoclonal antibodies.

Five monoclonal antibodies specific for the loop region of hen egg lysozyme were prepared by immunisation with a synthetic conjugate of a proteolytic fragment of lysozyme coupled to bovine serum albumin. Their fine specificities were investigated using a panel of variant lysozymes and peptide fragments of lysozyme in a quantitative radio-immunoassay procedure. Knowledge of the structure of hen lysozyme to high resolution and the use of computer graphics enables the localisation of the epitopes recognised by the antibodies with some precision. The antibodies were shown to define three distinct, overlapping epitopes within what was previously considered to be a single antigenic site. These results are discussed in relation to current ideas of the antigenic nature of proteins and other recent studies in which anti-protein antibodies have been elicited by immunisation with small peptides.     

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).