Enzymatic and immunochemical properties of lysozyme—Restriction of recognition of lysozyme antigenic determinants in pigs

Pigs immunized with lysozyme responded by producing only nonprecipitating antibody throughout the immunization period. Fig antilysozyme antibodies were found to be resistant to papain fragmentation, only 33% of the antibodies were fragmented with papain. From the binding of fluorescein labeled or ¹⁴C-labeled lysozyme to antilysozyme antibodies it was concluded that the antibodies elicited in pigs recognized only two antigenic determinants of lysozyme. These results were confirmed from the binding of Fab fragments to ¹⁴C-lysozyme. Fab fragments prepared from precipitating rabbit antilysozyme antibody bound ¹⁴C-lysozyme at a molar ratio of Fab/lysozyme = 3. Therefore nonprecipitating antibodies are the outcome of recognition of only two antigenic determinants on lysozyme and inability to form a lattice structure when antibody and antigen interact. This work emphasizes the limitations of using antibodies as a biological reagent for delineating the antigenic determinants on proteins.     

Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda

Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR.     

Computer modeling of electrostatic steering and orientational effects in antibody-antigen association.

Brownian dynamics simulations are performed to investigate the role of long-range electrostatic forces in the association of the monoclonal antibody HyHEL-5 with hen egg lysozyme. The electrostatic field of the antibody is obtained from a solution of the nonlinear Poisson-Boltzmann using the x-ray crystal coordinates of this protein. The lysozyme is represented as an asymmetric dumbell consisting of two spheres of unequal size, an arrangement that allows for the modeling of the orientational requirements for docking. Calculations are done with the wild-type antibody and several point mutants at different ionic strengths. Changes in the charge distribution of the lysozyme are also considered. Results are compared with experiment and a simpler model in which the lysozyme is approximately by a single charged sphere.     

Improvement of Human lysozyme Expression in Transgenic Rice Grain by Combining Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) <Emphasis Type="Italic">puroindoline b</Emphasis> and Rice <Emphasis Type="Italic">(Oryza sativa)</Emphasis> Gt1 Promoters and Signal Peptides

Heterologous protein expression levels in transgenic plants are of critical importance in the production of plant-made pharmaceuticals (PMPs). We studied a puroindoline b promoter and signal peptide (Tapur) driving human lysozyme expression in rice endosperm. The results demonstrated that human lysozyme expressed under the control of the Tapur cassette is seed-specific, readily extractable, active, and properly processed. Immuno-electron microscopy indicated that lysozyme expressed from this cassette is localized in protein bodies I and II in rice endosperm cells, demonstrating that this non-storage promoter and signal peptide can be used for targeting human lysozyme to rice protein bodies. We successfully employed a strategy to improve the expression of human lysozyme in transgenic rice grain by combining the Tapur cassette with our well established Gt1 expression system. The results demonstrated that when the two expression cassettes were combined, the expression level of human lysozyme increased from 5.24 ± 0.34 mg⁻¹ g flour for the best single cassette line to 9.24 ± 0.06 mg⁻¹ g flour in the best double cassette line, indicating an additive effect on expression of human lysozyme in rice grain.     

PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in Tetrahymena thermophila

Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone which functions in many mammalian systems, including the nervous and digestive systems. Using in vivo behavioral studies, we have found that this hormone functions as a chemorepellent in Tetrahymena thermophila with an EC₅₀ of 10 nM. Cells previously adapted to PACAP-38 were found to be adapted to lysozyme, and vice versa. Furthermore, the in vivo behavioral activity of PACAP-38 was blocked by addition of the anti-lysozyme receptor antibody, 5545. Chemorepellent activity of PACAP-38 was also inhibited by the addition of neomycin sulfate (inhibition constant K _i=0.080 μmol · l⁻¹), a competitive inhibitor of lysozyme binding to its receptor. PACAP-38 is a more potent and specific agonist for the lysozyme receptor than either intact lysozyme or CB2, a 24-amino acid fragment of lysozyme. Accepted: 11 October 1999     

Construction of affinity changeable antibody in response to Ca<Superscript>2+</Superscript>

Immunoaffinity chromatography is a powerful method for purification of proteins because of the high selectivity and avidity of antibodies. Due to the strength of antigen–antibody binding, however, elution of proteins bound to antibodies that are covalently immobilized on the column is performed by temporary denaturation of the antibody. Therefore, the development of milder elution conditions could improve the recovery of the antibodies and prolong the life of the immunoaffinity column. We describe the design and construction of an antibody that changes its affinity in response to external stimuli. The heavy chain and light chain of a single chain Fv of the D1.3 antibody against hen egg-white lysozyme (HEL) were fused at the N- and C-termini, respectively, of the calmodulin-M13 fusion protein. The affinity of this fusion protein for HEL could be modulated by changing the Ca²⁺ concentration.     

Backbone resonance assignments of monomeric SOD1 in dilute and crowded environments

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to movement disorders. In motor neurons of ALS patients, intracellular aggregates of superoxide dismutase 1 (SOD1) have often been observed. To elucidate the aggregation mechanism, it is important to analyze the folding equilibrium of SOD1 between folded and aggregation-prone unfolded states. However, in most cases, this folding equilibrium has been studied in dilute solution even though the aggregate formation occurs in a highly crowded intracellular environment. Indeed, a recent study reported that the folding stability of SOD1 decreased in an environment containing protein crowder molecules. To understand such a destabilization effect due to protein crowders, it is necessary to obtain more precise structural information on SOD1 in the presence of protein crowders. Here, we report the ¹H, ¹³C, and ¹⁵N backbone resonance assignments of monomeric SOD1 in the absence and presence of the protein crowder lysozyme. The chemical shift differences caused by addition of lysozyme suggest that SOD1 associated with lysozyme via negatively charged surfaces. Based on the assigned chemical shifts, the presence of lysozyme has a limited influence on the secondary structure of SOD1. We anticipate that our assignments will provide an important basis for elucidation of the crowding-induced folding destabilization of SOD1.     

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of N^α-[¹⁴C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of N^α-[¹⁴C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 10⁷ to 2.6 × 10⁸m^?1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.     

Mapping the antigenic epitope for a monoclonal antibody against lysozyme

A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of K?hler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds. The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody. Duck lysozyme did not react with the antibody at all. Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes. Arg 68 was identified as a determining amino acid. Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope. The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme. Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114. The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site. Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.     

Studies on lysozyme modifications induced by substituted p-benzoquinones

Protein misfolding can facilitate a protein damaging process and makes it susceptible to a series of events such as unfolding, adduct formation, oligomerization, or aggregation. Loss of a protein’s native structure may result in its biological malfunction and/or cellular toxicity that could cause associated diseases. Several factors were identified for causing structural changes of a protein, however quinone-induced protein modifications received very little attention whether for amyloidal or non-amyloidal proteins. In this paper, we report our investigation on lysozyme modifications upon treatment with selected benzoquinones (BQs), utilizing fluorescence spectroscopy including anisotropy determination, UV–Vis spectroscopy, and SDS-PAGE. Lysozyme was reacted with substituted BQs in order to examine substituent effects on protein modifications. In addition, we evaluated lysozyme modifications induced by 1,4-benzoquinone in concentration-, pH-, temperature-, and time-dependent studies. Our study shows that all BQs can readily modify lysozyme in a complex manner through adduct formation, oligomerization, polymeric aggregation, and/or fibrilization. Electrochemical properties of selected BQs were monitored using cyclic voltammetry in phosphate buffered aqueous solution, and it was found that quinone reduction potentials correlate well with their reactivity trend toward lysozyme.     

Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT^red) while oxidized dithiothreitol (DTT^ox) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT^red was added within 8 days of incubation. Our results showed that the initial addition of DTT^red interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT^red-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.     

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

Conservation of water molecules in an antibody–antigen interaction

The solvation of the antibody–antigen Fv D1.3–lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3–lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-abti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody–lysozyme complexes. The antibody–lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3–lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody–antigen interaction the solvent molecules fill many interface cavities. Comparison with x-ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Uo to 23 additional water molecules are also found in the antibody–antigen interface, however these waters do no bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody–antigen interface with the formation of the complex. While the current crystal models of the D1.3–lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well-ordered interface water contribute a net gain of 10 hydrogen bonds to complex stability.     

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme. Consequences of the replacement of Asp 101 by glycine

The nuclear magnetic resonance spectrum of the ¹⁹F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the ¹⁹F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK_a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β-d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.     

Crystallization,preliminary X-ray diffraction study,and crystal packing of a complex between anti-Hen lysozyme antibody F9.13.7 and Guinea-fowl lysozyme

The complex formed between the Fab fragment of a murine monoclonal anti-hen egg lysozyme antibody F9.13.7 and the het-erologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 Å resolution, belong to the monoclinic space group P2₁, with a = 83.7 Å, b = 195.5 Å, c = 50.2 Å, β = 108.5° and have two molecules of the complex in the asymmetric unit The three-dimensional structure has been determined from a preliminary data set to 4 Å using molecular replacement techniques. The lysozyme–Fab complexes are arranged with their long molecular axes approximately parallel to the crystallo-graphic unique axis. Fab F9.13.7 binds an anti-genie determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10. © 1993 Wiley-Liss, Inc.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Cell-mediated and humoral immunity in mice: cross reaction between lysozyme and S-carboxymethylated lysozyme studied by a modified footpad test.

The mouse sensitized by subcutaneous (sc) injection of lysozyme in emulsion of Freund's complete adjuvant (FCA) was shown by a modified footpad test to develop three kinds of hypersensitivities. Injecting lysozyme in 2.5-mul emulsion of Freund's incomplete adjuvant (FIA) into the footpad elicited strong footpad swelling in 30 min (anaphylactic reaction), in 3 hr (Arthus-type reaction) and in 24 hr (delayed-type hypersensitivity; DTH). The mice showing anaphylactic reaction in the footpad test manifested severe active systemic anaphylaxis, and the sera of these animals showed high IgG1 antibody titers with only sparingly detectable or no IgE antibody titers. In the sensitizing system with the use of FCA, the antigenicity of S-carboxymethylated lysozyme (CM-lysozyme) devoid of the three-dimensional conformation of lysozyme was compared with that of the native molecule. CM-lysozyme and lysozyme completely cross-reacted to each other in DTH, but not at all in the anaphylactic or Arthus-type reaction or in IgG1 antibody production. CM-lysozyme was shown also to have the ability to bestow immunological memory for the induction of humoral immunity against lysozyme; intravenous (iv) injection of lysozyme in saline or sc injection of CM-lysozyme-FCA alone failed to induce immediate hypersensitivities and IgG1 antibody production against lysozyme, but pre-sensitization by sc injection of CM-lysozyme-FCA enabled the animal to induce these responses to significant levels when iv injection of lysozyme in saline was given as a booster.     

Affinity dye–ligand poly(hydroxyethyl methacrylate)/chitosan composite membrane for adsorption lysozyme and kinetic properties

A composite membrane from 2-hydroxyethyl methacrylate (HEMA) and poly(hydroxyethyl methacrylate)/chitosan (pHEMA/chitosan) was synthesized via UV initiated photo-polymerization in the presence of an initiator α,α′-azoisobutyronitrile (AIBN). Procion Brown MX 5BR was then covalently immobilized onto composite membrane as a dye–ligand. The binding characteristics of a model protein (i.e. lysozyme) to the dye–ligand immobilized affinity membrane have been investigated from aqueous solution using the plain composite membrane as a control system. The experimental data was analyzed using two adsorption kinetic models, the pseudo-first-order and the pseudo-second-order, to determine the best-fit equation for the adsorption of lysozyme onto affinity composite membrane. The second-order equation for the adsorption of lysozyme on the dye–ligand membrane systems is the most appropriate equation to predict the adsorption capacity for the affinity membrane. The reversible lysozyme adsorption on the affinity membrane obeyed the Freundlich isotherm. The lysozyme adsorption capacity of the plain membrane and the dye–ligand affinity membrane were 8.3 and 121.5 mg ml⁻¹, respectively.     

Short-range conformational energies,secondary structure propensities,and recognition of correct sequence-structure matches

A statistical analysis of known structures is made for an assessment of the utility of short-range energy considerations. For each type of amino acid, the potentials governing (1) the torsions and bond angle changes of virtual C^α-C^α bonds and (2) the coupling between torsion and bond angle changes are derived. These contribute approximately −2 RT per residue to the stability of native proteins, approximately half of which is due to coupling effects. The torsional potentials for the α-helical states of different residues are verified to be strongly correlated with the free-energy change measurements made upon single-site mutations at solvent-exposed regions. Likewise, a satisfactory correlation is shown between the β-sheet potentials of different amino acids and the scales from free-energy measurements, despite the role of tertiary context in stabilizing β-sheets. Furthermore, there is excellent agreement between our residue-specific potentials for α-helical state and other thermodynamic based scales. Threading experiments performed by using an inverse folding protocol show that 50 of 62 test structures correctly recognize their native sequence on the basis of short-range potentials. The performance is improved to 55, upon simultaneous consideration of short-range potentials and the nonbonded interaction potentials between sequentially distant residues. Interactions between near residues along the primary structure, i.e., the local or short-range interactions, are known to be insufficient, alone, for understanding the tertiary structural preferences of proteins alone. Yet, knowledge of short-range conformational potentials permits rationalizing the secondary structure propensities and aids in the discrimination between correct and incorrect tertiary folds. Proteins 29:292–308, 1997. © 1997 Wiley-Liss, Inc.