Avian Lysozymes Research Articles

Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity

Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40’s ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS.

Bare-Faced Curassow Lysozyme Carrying Amino Acid Substitutions at Subsites E and F Shows a Change in Activity against Chitooligosaccharide Caused by a Local Conformational Change

A new form of avian lysozyme, bare-faced curassow lysozyme (BCL), was purified and chemically sequenced. Of the 26 substitutions relative to chicken lysozyme, three, F34Y, T47S, and R114H, are of substrate-interacting residues in the E and F subsites, which would contribute to the acceptor binding for transglycosylation. T47S is a novel substitution in this lysozyme class. While other lysozymes also have substitutions at positions 114 and 34, they also contain numerous others, including ones in the other substrate binding sites, A-D. Furthermore, T47S lies on the left side, while F34Y and R114H are located on the right side of the E-F subsites. BCL therefore should allow comparison of the independent contributions of these sites to substrate binding and transglycosylation. The activity toward the N-acetylglucosamine pentamer revealed that the substitutions at the E-F sites reduced the binding free energies at the E-F sites and the rate constant for transglycosylation without the conformation change of other substrate binding sites on the protein. MD simulation analysis of BCL suggested that the substituted amino acids changed the local conformation of this lysozyme at the E-F sites.

Cross‐reactivity and Heat Lability of Antigenic Determinants of Duck and Goose Lysozymes

ABSTRACTTwo panels of monoclonal antibodies (Mabs) raised against duck (Barbary) egg white lysozyme or hen egg white lysozyme, were tested in antigen‐coated plate (ACP) and double antibody sandwich (DAS) ELISA for cross‐reaction with various avian lysozymes. The antibodies to hen lysozyme cross‐reacted with goose lysozyme, but most antibodies to duck lysozyme reacted with it. One antibody to duck lysozyme reacted more strongly with goose lysozyme than with the homologous antigen, a heterospecific reaction confirmed by biosensor technology. Many lysozyme epitopes recognized by different antibodies showed considerable resistance to heat denaturation. Such Mabs may be useful for detecting chicken liver adulterants in “foies gras” of goose or duck origin.

Antibody Response of Murine B1 Cells to Hen Eggwhite Lysozyme

Cell transfer studies were performed to investigate the ability of murine peritoneal B1 cells to produce specific IgG antibody against the T-dependent protein antigen, hen eggwhite lysozyme (HEL). Peritoneal cells (PeC) from normal BALB/c mice were transferred into newborn, allotype-congenic, C.B-17 severe combined immunodeficient (SCID) mice alone or together with splenic T cells from HEL-primed C.B-17 mice. After immunization with HEL, only those mice that received both PeC and primed T cells produced HEL-specific IgG of the PeC donor allotype. To identify the B cell subset responsible for antibody production, PeC were sorted before transfer into B1 and conventional B (B2) cell populations. It was found that transfer for purified B1 cells plus primed T cells resulted in high levels of IgG1 anti-HEL, in approximately half of the SCID recipients, while mice receiving B2 cells produced little detectable antibody. The responses consisted primarily of IgG1κ anti-HEL, with no significant IgM or λ-bearing components. Seventeen HEL-specific hybridomas of BALB/c origin, i.e., derived from the B1 cell donor, were obtained from reconstituted SCID mice after various periods of immunization and analyzed for fine specificity using a panel of avian lysozymes. All but one of the B1 cell-derived mAbs recognized an HEL epitope not present on the closely related bobwhite quail lysozyme (differing from HEL at only 4 of 129 amino acid positions). While IEF analyses demonstrated the presence of extensive clonotypic diversity, the epitope specificity pattern, which is rare among B2-cell-derived antibodies, suggests that the B1 cell recognition repertoire for HEL is severely limited.

Structure of an Antibody–Lysozyme Complex Unexpected Effect of a Conservative Mutation

The structure of the complex between the Fab HyHEL-5 and chicken lysozyme revealed a large interface region containing 23 lysozyme and 28 Fab residues. Arg68 of the lysozyme is centrally placed in this interface and theoretical studies together with binding assays of this Fab to different avian lysozymes have previously shown that this arginine residues is an important contributor to the binding. The Arg69*rarr;Lys mutant binds 10 3times less well to the HyHEL-5 Fab. We have examined the refined crystal structure of the complex of this mutant lysozyme with the Fab. No global changes occur, but there is an introduction of a new water molecule into the interface that mediates the hydrogen bonding interactions between the lysine and residues on the Fab. These data are compared with the effects of similar changes on the inhibition of serine proteases such as trypsin where the energetic effects of this substitution are small.

Sequences of Two Highly Divergent Canine Type c Lysozymes: Implications for the Evolutionary Origins of the Lysozyme/α-Lactalbumin Superfamily

The amino acid sequences of two canine lysozymes, from milk and spleen, have been elucidated by direct sequence analyses of the purified proteins and fragments generated from them. The two enzymes are highly divergent, differing from each other by 45% in sequence, but each is closely similar to lysozymes previously obtained from other mammalian species. The milk lysozyme is similar in sequence to equine and donkey milk lysozymes (83% identity) and, like these enzymes, contains a bound Ca2+ ion while the spleen enzyme is most similar in sequence to the majority of previously studied mammalian and avian lysozymes (80 to 83% identity) and, based on its sequence, does not contain a Ca2+-binding site. This demonstrates that Ca2+-binding lysozymes are expressed in at least two mammalian orders, the carnivores and perissodactyls, as well as confirming that the genes for the Ca2+-binding and conventional lysozymes are paralogous. The latter point was further confirmed by the isolation and partial sequence analysis of a conventional lysozyme from equine spleen. The relationships of these new lysozyme sequences to those of other lysozymes and their homologues, the α-lactalbumins, were analyzed using different molecular phylogeny algorithms, producing a new model for the evolutionary origins of the superfamily. The most significant conclusion to be drawn from this model is that Ca2+-binding activity was an ancient feature of this protein superfamily which was lost during the evolutionary development of the conventional lysozymes. It also supports a previous suggestion that the α-lactalbumins and lysozymes diverged at a time earlier than the divergence of the fishes and tetrapods.

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.

Characteristics of the epitope of protein-reactive anti-peptide antibodies.

The specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys64-Cys80 and Cys76-Cys94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.

Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.

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.

The effect of protein contaminants on the crystallization of turkey egg white lysozyme

We report here a series of studies on the controlled contamination of crystallizing solutions of the hexagonal form of turkey egg white lysozyme (TEWL) carried out to understand the effects of impurities on the nucleation and growth of protein crystals. The contamination of TEWL solutions with any of three other avian lysozymes affects both the nucleation and the growth processes. For hen and quail egg white lysozymes, low and medium levels of contamination result in partial inhibition of nucleation and shortening of the c-axis. Further increase of the contaminant concentration leads to detectable co-crystallization. A different effect is obtained when using the pheasant egg white lysozyme. Contamination by an unrelated protein, ribonuclease A, has an effect on the nucleation levels that is similar to those observed with the avian lysozymes. However, no effect on TEWL crystal morphology is observed. Thus, in the case of TEWL crystals, one can distinguish between a specific effect on crystal morphology induced by related proteins and a more general inhibitory effect on the nucleation levels observed in all cases studied here.

Three-dimensional structure and antigen binding specificity of antibodies

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigen determinant with the antibody combining site.

Preliminary crystallographic examination of a novel fungal lysozyme from Chalaropsis.

The lysozyme from the fungus of the Chalaropsis species has been crystallized. This lysozyme (Mr 22,415) displays no sequence homology with avian, phage, or mammalian lysozymes, however, preliminary studies indicate significant sequence homology with the bacterial lysozyme from Streptomyces. Both enzymes are unusual in possessing beta-1,4-N-acetylmuramidase and beta-1,4-N,6-O-diacetylmuramidase activity. The crystals grow from solutions of ammonium sulfate during growth periods from several months to a year. The space group is P2(1)2(1)2(1) with a = 34.0 A, b = 42.6 A, c = 122.1 A. Preliminary data indicate that there is 1 molecule/asymmetric unit. A complete native data set has been collected to 2.57-A resolution. The crystals are highly ordered and exhibit diffraction patterns to d-spacings less than 1.5 A.

Chromatographic resolution of lysozyme variants

There are seven avian lysozyme variants of nearly identical three-dimensional structure which have amino acid substitutions broadly distributed on their surface. By using these protein variants, it was possible to study the relationship between protein structure and chromatographic retention. It was determined that according to the mode of separation various regions of the proteins surface determine chromatographic retention. At one extreme, immunosorbents targeted a very small region on the protein surface. Hydrophobic interaction chromatography was an intermediate case in which one surface domain of the lysozymes controlled chromatographic behavior. At the opposite extreme, cation-exchange columns probed most of the protein surface. It was concluded that identification of random variations in protein structure will be most successfully detected by a separation mode that broadly targets the surface of a protein.

Antigen specificity and cross-reactivity of monoclonal anti-lysozyme antibodies

Twenty-seven murine monoclonal anti-hen egg-white lysozyme (HEL) antibodies were tested with a panel of nine antigens: eight avian lysozymes and human lysozyme. The antibodies were arranged into 10 groups based on their antigen specificity and cross-reactivity. Antigenic determinants recognized by each group of antibodies were tentatively identified. They are located at different points of the HEL accessible surface in agreement with the notion that its entire surface has an antigenic potential. The affinity constants of antibodies representative of seven of the 10 groups range from 0.79 × 10 7 to 5.3 × 10 7 M −1. Heteroclitic antibodies occurring in some of the groups bind heterologous lysozymes with somewhat higher association constants than those for the homologous antigen (HEL). Their broader specificities do not correlate with overall lower association constants, but rather with the occurrence of public epitopes in the panel of avian lysozymes. Although the reaction of two antibodies with the solid-phase coupled antigen is not always additive, detailed interpretation of these results and the observation of actual ternary complexes Fab-HEL-Fab rules out the occurrence of conformational changes in the complexed antigen.

Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozyme.

The epitopes recognized by six new BALB/c hybridomas specific for the protein antigen hen egg-white lysozyme (HEL) were mapped in detail. Although fine specificities of all the antibodies were distinct, many of the epitopes overlap in complex patterns. The antibodies could be grouped into three complementation groups, one of which also included the previously characterized HyHEL -5 which is specific for Arg68 . Complex interactions were observed among the antibodies, both among and within complementation groups, including nonreciprocal competition and enhanced binding. Two of the complementation groups mapped near the catalytic site in a new antigenic region. The antibodies HyHEL -8 and HyHEL -10 had very similar and over-lapping specificities, and may recognize very closely related epitopes. The results suggest that the epitopes may form a continuous antigenic surface, and that antigenic regions correspond to structural domains defined by the tertiary structure of HEL.

VL-VH expression by monoclonal antibodies recognizing avian lysozyme.

Seven BALB/c hybridoma antibodies directed against the protein antigen, hen egg-white lysozyme c (HEL), were characterized on the basis of their ability to bind lysozymes from 10 species of birds, and their ability to bind HEL competitively. The hybridomas were separable into three complementation groups based upon competitive interactions. The fine specificities of all antibodies were distinct, but two, HyHEL-8 and HyHEL-10, had very similar and overlapping reactivity patterns. To test the hypothesis that VL-VH pairing correlates with binding specificity, the N-terminal amino acid sequences were determined to identify the VL and VH isotopes (subgroups) of the anti-HEL antibodies. HyHEL-8 and -10 shared the VK23 light chain isotype and nearly identical heavy chains in Kabat subgroup I, whereas the heavy and light chain isotypes of all other antibodies differed from HyHEL-8 and -10 and from each other. The heavy and light chain isotypes expressed by HyHEL-8 and -10 are also expressed by XRPC-25, a DNP-binding myeloma protein that does not bind lysozyme. These results are discussed with respect to the contributions of various genetic sources of structural diversity to antibody functional diversity.

A monoclonal antibody specific for a distinct region of hen egg-white lysozyme

Six stable hybrid cell lines that secrete homogeneous anti-hen egg-white lysozyme (HEL) antibodies were prepared by cell fusion between cells of immunoglobulin non-synthetizing myeloma (SP2/0-Ag14) and spleens of A/J mice. One (Hyb · Cl) of these hybridoma antibodies was found to be specific for the N- and C-terminal portions of HEL. It showed restricted heterogeneity on isoelectrofocusing and an isoelectric point of about pH 6.0. The heavy- and light-chain classes of the Hyb · Cl antibody were γ 1 and kappa, respectively. The association constant of the Hyb · Cl antibody with HEL at 37°C was 3.3 × 10 7 ( L/ M). The Hyb · Cl antibody was further characterized by its cross reactivities with other avian lysozymes. The association constants ( K I s) of cross-reactive avian egg-white lysozymes at 37°C, calculated from the inhibitions of binding of 125I-HEL to Hyb · Cl antibody were 3.2 × 10 7 ( L/ M) for both Japanese quail lysozyme (JEL) and turkey lysozyme (TEL) and 1.2 × 10 6 ( L/M) for two duck lysozymes (DL-1 and DL-2). The partial N-terminal amino acid sequences of the heavy and light chains of Hyb · Cl antibody up to the 25th residues were also determined.

A predominant idiotype on anti-hen egg white lysozyme antibodies from diverse mouses strains.

A predominant idiotype was detected on almost all antibody molecules directed against the multideterminant antigen, hen egg white lysozyme (HEL). This idiotypic set was defined by rabbit and quinea pig antiidiotypic antisera raised against a rare cross-reactive antibody population isolated and from a heterogeneous BALB/c anti-human lysozyme (HUL) humoral response. This isiotype was fond on the majority of all HEL-reactive antibody from all strain tested; its presence was independent of Ig-1 allotype of H-2 haplotype. The idiotype was also present on the majority of anti-HEL antibody from nonresponder mice immunized with keyhole limpet hemocyanin coupled to HEL. The idiotype was not found on normal mouse immunoglobulin or on antibodies specific to two other proteins, HUL and ribonuclease, which are similar to HEl in size and charge. Individual idiotype-positive anti-HEL antibody populations exhibited specificity differences when analyzed for reactivity toward other avian lysozymes, suggesting that distinct clonal products shared the idiotype specificities. Fractionation of heterogeneous anti-HEL antibodies by preparative isoelectric focusing yielded distinct antibody fractions that were demonstrated to share the relevant idiotype determinants. The data are discussed in terms of the HEL peptide specificites of idiotype-positive and -negative antibodies.

Amino acid sequence and immunological properties of chalchalaca egg white lysozyme.

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 2 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 chachalaca lysozyme 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.