Goose Lysozyme Research Articles

Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis

Lysozyme is an important defense molecule of the innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of b-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of four g-type lysozymes were identified in Ciona intestinalis. Phylogenetic analysis and modelling supported the hypothesis of a close relationship with the vertebrate g-type lysozymes suggesting that the C. intestinalis g-type lysozyme genes (CiLys-g1, Cilys-g2, CiLys-g3, CiLys-g4) share a common ancestor in the chordate lineage. Protein motif searches indicated that C. intestinalis g-type lysozymes contain a GEWL domain with a GXXQ signature, typical of goose lysozymes. Quantitative Real-Time PCR analysis results showed that transcripts are expressed in various tissues from C. intestinalis. In order to determine the involvement of C. intestinalis g-type lysozymes in immunity, their expression was analyzed in the pharynx, showing that transcripts were significantly up-regulated in response to a challenge with lipopolysaccharide (LPS). These data support the view that CiLys g-type are molecules with potential for immune defense system against bacterial infection.

Amino Acid Sequence of Egyptian Goose Egg-White Lysozyme and Effects of Amino Acid Substitution on the Enzymatic Activity

The amino acid sequence of Egyptian goose lysozyme (EGL) from egg-white and its enzymatic properties were analyzed. The established sequence had the highest similarity to wood duck lysozyme (WDL) with five amino acid substitutions, and had eighteen substitutions difference from hen egg-white lysozyme (HEL). Tyr34 and Gly37 were found at subsites E and F of the active site when compared with HEL. The experimental time-course characteristics of EGL against the N-acetylglucosamine pentamer substrate, (GlcNAc)(5), revealed higher production of (GlcNAc)(4) and lower production of (GlcNAc)(2) when compared with HEL. The saccharide-binding ability of subsites A-C in EGL was also found to be weaker than in HEL. An analysis of the enzymatic reactions of five mutants in respect of positions 34, 37 and 71 in HEL indicated the time-course characteristics of EGL to be caused by the combination of three substitutions (F34Y, N37G and G71R) between HEL and EGL. A computer simulation of the EGL-catalyzed reaction suggested that the time-course characteristics of EGL resulted from the difference in the binding free energy for subsites A, B, E and F and the rate constant of transglycosylation between EGL and HEL.

Bacillus subtilis CwlQ (previous YjbJ) is a bifunctional enzyme exhibiting muramidase and soluble-lytic transglycosylase activities

CwlQ (previous YjbJ) is one of the putative cell wall hydrolases in Bacillus subtilis. Its domain has an amino acid sequence similar to the soluble-lytic transglycosylase (SLT) of Escherichia coli Slt70 and also goose lysozyme (muramidase). To characterize the enzyme, the domain of CwlQ was cloned and expressed in E. coli. The purified CwlQ protein exhibited cell wall hydrolytic activity. Surprisingly, RP-HPLC, mass spectrometry (MS), and MS/MS analyses showed that CwlQ produces two products, 1,6-anhydro- N-acetylmuramic acid and N-acetylmuramic acid, thus indicating that CwlQ is a bifunctional enzyme. The site-directed mutagenesis revealed that glutamic acid 85 (Glu-85) is an amino acid residue essential to both activities.

Antimicrobial peptides derived from goose egg white lysozyme

Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.

Identification and Characterization of Novel Cell Wall Hydrolase CwlT: A TWO-DOMAIN AUTOLYSIN EXHIBITING N-ACETYLMURAMIDASE AND dl-ENDOPEPTIDASE ACTIVITIES*

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."

Enzymatic Properties of Rhea Lysozyme

Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.

Sweetness and enzymatic activity of lysozyme.

Hen egg lysozyme elicits a sweet taste sensation for human beings. Effects of reduction of disulfide bonds, heat treatment, and chemical modification of hen egg lysozyme on both sweetness and hydrolytic activity were investigated. Both the sweetness and enzymatic activities were lost when the intradisulfide linkage in a lysozyme molecule was reduced and S-3-(trimethylated amino) propylated. The sweetness and enzymatic activity of lysozyme were lost on heating at 95 degrees C for 18 h. These facts suggest that tertiary structures of lysozyme are indispensable for eliciting a sweet taste as well as enzymatic activity. Although the modification of carboxyl residues in a lysozyme by glycine methylester or aminomethansulfonic acid resulted in the loss of enzymatic activity by blocking the catalytic residues, the sweetness was fully retained. These results indicate that the sweetness of lysozyme was independent of its enzymatic activity. The lysozyme purified from goose egg white similarly elicited a sweet taste, although goose (g-type) lysozyme is quite different from hen egg lysozyme (c-type) on the basis of structural, immunological, and enzymatic properties. These findings indicate that a specific protein property of lysozyme is required for sweetness elicitation and that the enzymatic activity and carbohydrates produced by enzymatic reaction are not related to the sweet taste.

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white.

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.

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.

The refined structures of goose lysozyme and its complex with a bound trisaccharide show that the “Goose-type” ly'sozymes lack a catalytic aspartate residue

The structure of goose egg-white lysozyme (GEWL) has been refined to an R-value of 15.9% at 1.6 A resolution. Details of the structure determination, the refinement and the structure itself are presented. The structure of a complex of the enzyme with the trisaccharide of N-acetyl glucosamine has also been determined and refined at 1.6 A resolution. The trisaccharide occupies sites analogous to the B, C and D subsites of chicken (HEWL) and phage T4 (T4L) lysozymes. All three lysozymes (GEWL, HEWL and T4L) display the same characteristic set of bridging hydrogen bonds between backbone atoms of the protein and the 2-acetamido group of the saccharide in subsite C. Glu73 of GEWL is seen to correspond closely to Glu35 of HEWL (and to Glu11 of T4L) and supports the established view that this group is critically involved in the catalytic mechanism. There is, however, no obvious residue in goose lysozyme that is a counterpart of Asp52 of chicken lysozyme (or of Asp20 in T4L), suggesting that a second acidic residue is not essential for the catalytic activity of goose lysozyme, and may not be required for the activity of other lysozymes.

Lysozymes' esterase activity

A non-specific esterase activity of hen lysozyme (c-type) has previously been described: histidine was claimed to be involved in this activity. The present letter reports that a histidine-less lysozyme from the duck egg-white (c-type) as well as a goose lysozyme (g-type) possess such an esterase activity determined using p-nitrophenylacetate.

Complete amino acid sequence of Embden goose ( Anser anser) egg-white lysozyme

The complete amino acid sequence of Embden goose ( Anser anser) egg-white lysozyme (EC 3.2.1.17) has been proposed. The structure was established by comparing the citraconylated tryptic peptides of Embden goose lysozyme with the corresponding peptides from the black swan goose-type lysozyme. Those peptides which differed in amino acid composition were subjected to sequence analysis; the alignment of the tryptic peptides was achieved by analogy with other goose-type lysozymes. Embden goose lysozyme is a single polypeptide chain of 185 amino acids. It closely resembles the goose-type lysozymes from the black swan and the ostrich, differing in 6 and 30 residues, respectively.

Comparative studies of lysozymes at the level of their tryptophan residues

1. 1. Lysozymes from hen, duck II and goose egg white and human lysozyme were compared at the level of their tryptophyl residues using the solvent perturbation technique and fluorescence emission in addition to two chemical modifications. 2. 2. The three tryptophyl residues of goose lysozyme were found completely exposed to the solvent. 3. 3. The study of the situation of the aromatic residues of hen, duck II and human lysozymes provided additional evidence that the three dimensional structures of these enzymes are very similar although an unexpected reactivity of tryptophan 28 of human lysozyme towards N-bromosuccinimide could be evidenced.

Multiple Genes for Lysozyme in Birds: Studies on Black Swan Egg White Lysozymes

In contrast to other bird species that have been studied, not one but two distinct lysozymes have been isolated from the egg white of the black swan (Cygnus atratus). One is similar in molecular weight, NH2-terminal amino acid sequence, amino acid composition, specific activity, and immunological cross-reactivity to the lysozymes found in the egg white of the chicken, duck, and many other birds. The other black swan lysozyme shows no resemblance to these “chick-type” enzymes by the above criteria. It is very similar to the lysozyme found until now only in the egg white of the Embden goose. This black swan lysozyme and Embden goose lysozyme have a molecular weight of about 20,500 and are identical in the sequence of their first 20 NH2-terminal amino acids and in immunological cross-reactivity. Amino acid composition data indicate that these two “goose-type” lysozymes differ in sequence by a minimum of four amino acid substitutions. The differences in the structures of the chick-type and goose-type lysozymes suggest that they are products of two distinct genetic loci. Some species contain only chick-type lysozymes in their egg white (e.g. chicken, duck), others only goose-type lysozymes (Embden goose), while others contain both types of enzyme (black swan). In the light of other evidence, this suggests that the genetic loci coding for both enzymes are present in all species and that the presence of either or both lysozymes in egg white is controlled by a species-specific regulatory mechanism.

Chemical Studies on the Enzymatic Specificity of Goose Egg White Lysozyme

In comparison with hen egg white lysozyme, goose lysozyme is known to be aberrant in both its structure and its enzymatic behavior in the presence of polymers of N-acetylglucosamine or cell suspensions of Micrococcus luteus. Our chemical studies show, however, that like the hen enzyme, goose lysozyme has muramidase activity. At several pH levels ranging from 3.5 to 7.1 the goose enzyme liberated the reducing ends of N-acetylmuramic acid residues in purified preparations of Escherichia coli and M. luteus peptidoglycans. In contrast to what is known about hen lysozyme, however, our results suggest that the goose enzyme has a distinct preference for N-acetylmuramic acid residues which are substituted with a peptide moiety. This difference in specificity towards the peptide portion of the peptidoglycan may be related to the biological function of lysozyme.

Multiple genes for lysozyme in birds

Quantitative microcomplement-fixation and immunodiffusion studies using antisera prepared against purified chicken and Embden goose lysozymes indicate that two lysozymes are present in black swan ( Cygnus atratus) egg white. Their different antigenic structures suggest that these two forms of lysozyme may be the products of nonallelic genes. The possibility of multiple gene loci for lysozyme or other proteins calls for caution in the interpretation of data on the rate of protein evolution.

Molecular palaeogenetics: Amino acid sequence homology in ribonuclease and lysozyme

1. 1. Three approaches are employed, together with appropriate statistical methods, to test the hypothesis that bovine pancreatic ribonuclease and chicken egg-white lysozyme are homologous proteins. 2. 2. All three methods (which are partially correlated) give probabilities ranging from 0·01 to 0·0008 that such a set of similarities could have arisen entirely at random. 3. 3. About 10 per cent of the original “protogene” remains as a “molecular fossil” in the desoxyribonucleotide sequence coding lysozyme and RNAse. 4. 4. Several pieces of evidence, including a curious heterogeneity in the purine-pyrimidine distribution between the first two and the third nucleotides in the messenger codon equivalents, suggest that the divergence of RNAse and lysozyme took place very early in the evolution of living organisms, perhaps near the origin of life itself. 5. 5. A possible homology exists between a decapeptide sequence of 'phage T-4 lysozyme and hen ovolysozyme. 6. 6. The peculiar amino acid composition of goose ovolysozyme can be explained by postulating a dirty deletion in the middle of the ancestral anserine lysozyme cistron; this has resulted in a single-letter coding frame shift, which “scrambles” the C-terminal half of goose lysozyme.