The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

Purification, amino acid sequence, and some properties of rabbit kidney lysozyme

The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.     

Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications to molecular evolution

The crystal structure of an antibacterial protein of immune origin (TSWAB), purified from tasar silkworm (Antheraea mylitta) larvae after induction by Escherichia coli infection, has been determined. This is the first insect lysozyme structure and represents induced lysozymes of innate immunity. The core structure of TSWAB is similar to c-type lysozymes and alpha-lactalbumins. However, TSWAB shows significant differences with respect to the other two proteins in the exposed loop regions. The catalytic residues in TSWAB are conserved with respect to the chicken lysozyme, indicating a common mechanism of action. However, differences in the noncatalytic residues in the substrate binding groove imply subtle differences in the specificity and the level of activity. Thus, conformational differences between TSWAB and chicken lysozyme exist, whereas functional mechanisms appear to be similar. On the other hand, alpha-lactalbumins and c-type lysozymes exhibit drastically different functions with conserved molecular conformation. It is evident that a common molecular scaffold is exploited in the three enzymes for apparently different physiological roles. It can be inferred on the basis of the structure-function comparison of these three proteins having common phylogenetic origin that the conformational changes in a protein are minimal during rapid evolution as compared with those in the normal course of evolution.     

Structure of the pigeon lysozyme and its relationship with other type c lysozymes

1. The secondary structure of the pigeon egg-white lysozyme shows important differences when compared to other type c lysozymes. These differences are mainly located at the region comprising residues 77-84. This segment contains one alpha-helix in the lysozymes c studied by means of an X-ray analysis, while the residues at such positions in pigeon lysozyme would form two beta-bends. 2. Analysis of the tertiary structure of the pigeon lysozyme by means of hydropathy profiles reveals that the above segment seems to be more hydrophilic in the pigeon enzyme than in other type c lysozymes. 3. Though a certain similarity to the calcium-binding loop of alpha-lactalbumins is detected in pigeon lysozyme, the circular dichroism spectra of the protein at neutral pH do not change in the presence of Ca2+ ions. 4. The presented structural analysis is discussed in terms of function-structure and antigenicity relationships between the type c lysozymes.     

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.     

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.     

Expression and purification of Artogeia rapae lysozyme II cDNA in a baculovirus/insect cell system

Previously, the Artogeia rapae lysozyme II (ARLII) gene was isolated and its complete nucleotide sequence was determined by RACE‐PCR from fat body of larvae injected with Escherichia coli. In the present study, the ARLII gene was expressed by using a baculovirus expression vector system (BEVS). The expression level of recombinant (r)ARLII protein was optimized by varying virus titer and time‐course of infection. The optimum protein expression conditions were infection of the cells at a multiplicity of infection of 10, and harvest at 84 h post‐infection. Under these conditions, we estimated the amount of rARLII produced in the BEVS to be 10 mg/mL. rARLII was purified from cell‐conditioned media using cation exchange column and reversed‐phase FPLC methods. Purified rARLII was able to form a clear zone in a lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 1.53 times higher than that of hen egg white lysozyme (HEWLZ) under the same conditions. The optimum temperature for the lytic activity of the rARLII was 50°C, and its temperature dependency was greater than that of HEWLZ at low temperatures (<65°C).     

Analysis of the internal motion of free and ligand-bound human lysozyme by use of 15N NMR relaxation measurement: a comparison with those of hen lysozyme

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3-bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari-Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al.. 1999, J Mol Biol 286:1547-1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3-bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand-bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate-binding mode or efficiency of activity between them.     

Comparison of goose-type, chicken-type, and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) egg-white lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983). In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the global and the local level. As in the globins, there are corresponding alpha-helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another. Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive alpha-helices in GEWL and three consecutive alpha-helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices. There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 A for Asp 86 and 4 A for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of the three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

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.     

Purification, characterization and comparison of reptile lysozymes

Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle (Trionyx sinensis), ASTL from Asiatic soft shelled turtle (Amyda cartilagenea) and GSTL from green sea turtle (Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 degrees C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila.     

Purification and characterization of bacteriophage 9NA lysozyme

Bacteriophage 9NA is a virulent phage of Salmonella typhimurium which induces a lysozyme in host cells toward the later stages of its multiplication. 9NA lysozyme has been purified about 1000 fold starting from the lysate of 9NA infected cells. The enzyme has an optimum pH between 7 and 8 and its activity is dependent on the ionic strength of the assay medium. Salts like NaCl and KCl are inhibitory to the lysozyme. Gram-negative cells act as better substrate for the lysozyme than do Gram-positive cells. The enzyme has a molecular weight of about 2.1 X 10(4) and rapidly loses its activity at temperatures higher than 45 degrees C. The properties of 9NA lysozyme have been compared with those of T4, lambda and P22 lysozymes.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

Functional relationships and structural determinants of two bacteriophage T4 lysozymes: a soluble (gene e) and a baseplate-associated (gene 5) protein

Lysozymes have proved useful for analyzing the relation between protein structure and function and evolution. In bacteriophage T4, the major soluble lysozyme is the product of the e gene, gpe (gene product = gp). This lysozyme destroys the wall of its host, Escherichia coli, at the end of infection to release progeny particles. Phage T4 contains two additional lysozymes that facilitate penetration of the baseplates into host cell walls during adsorption. At least one of these, a 44-kD protein, is encoded by gene 5. We show here that a segment of the gp5 lysozyme amino acid sequence, deduced from the DNA sequence of gene 5, is remarkably similar to that of the T4 gene e lysozyme. Both T4 lysozymes are somewhat similar to the lysozyme of the Salmonella phage P22, but there is little significant DNA sequence homology among the two T4 lysozyme genes and the P22 lysozyme gene. We speculate that these lysozymes are adapted to differences in the composition of the cell walls of E. coli and S. typhimurium. The cloned gene 5 of the phage T4 directs synthesis of a 63-kD precursor protein that is approximately 19 kD larger than the gene 5 protein isolated from baseplates. Gp5 first associates with gp26 to form the central hub of this structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of phage P22 gene 19 lysozyme inferred from its homology with phage T4 lysozyme. Implications for lysozyme evolution

The amino acid sequence of the lysozyme from phage P22 is shown to be homologous (26% identity) with the lysozyme from bacteriophage T4. The sequence correspondence suggests that the structure of P22 lysozyme is similar to the known structure of T4 lysozyme within the "core" of the molecule, including the active site cleft. However, P22 lysozyme appears to lack two surface loops present in T4 lysozyme. It is possible that P22 lysozyme may provide an "evolutionary link" between the phage-type lysozymes and the goose-type lysozymes.     

Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft

Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.     

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.     

Protein dissection experiments reveal key differences in the equilibrium folding of alpha-lactalbumin and the calcium binding lysozymes

The alpha-lactalbumins and c-type lysozymes have virtually identical structure but exhibit very different folding behavior. All alpha-lactalbumins form a well populated molten globule state, while most of the lysozymes do not. alpha-Lactalbumin consists of two subdomains, and the alpha-subdomain is considerably more structured in the molten globule state than the beta-subdomain. Constructs derived from the alpha-subdomain of human alpha-lactalbumin containing the A, B, D, and 3(10) helices are known to form a molten globule state in the absence of the rest of the protein (Demarest, S. et al. (1999) J. Mol. Biol. 294, 213-221). Here we reported comparative studies of constructs derived from the same regions of canine and equine lysozymes. These proteins form two of the most stable molten globule states among all the lysozymes. A construct containing the A, B, D, and 3(10) helices of equine lysozyme is partially helical but is less structured than the corresponding human alpha-lactalbumin peptide. Addition of the C-helix leads to a construct that is still less structured and less stable than the alpha-lactalbumin construct. The corresponding construct from canine lysozyme is also less structured and less stable than the alpha-lactalbumin peptide. Thus, molten globule formation in human alpha-lactalbumin can be driven by the isolated alpha-subdomain, while more extensive interactions are required to generate a stable molten globule in the two lysozymes. The stability of the canine and equine lysozyme constructs is similar, indicating that the extraordinary stability of the canine lysozyme molten globule is not due to an unusually stable isolated alpha-subdomain.     

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering.

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Lysozyme activity in animal extracts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Lysozyme activity was detected after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing 0.2% (W/V) autoclaved Micrococcus lysodeikticus cells as substrate. 2. Lysozyme activity appeared as clear lysis zones after incubation of opaque gels at 37 degrees C in buffered Triton X-100. 3. As low as 0.1 pg of purified hen egg white lysozyme could be detected after 16 hr incubation at pH 6.5. 4. Bands with lytic activity from kidney and pancreas acetone powders, bird's egg whites and vitelline membranes, animal sera and human saliva corresponded to c-type (Mr 14,500), g-type (Mr 20,500) or both lysozymes as far as molecular weight is concerned. 5. Some extracts, like porcine kidney, exhibited more than two bands. 6. Bands with lytic activity migrating at the level of g-type lysozymes were detected in some kidney and pancreas extracts.