Characterization of a novel lysozyme-like 4 gene in the rat

Lysozyme-like proteins (LYZLs) belong to the class of c-type lysozymes and are not well characterized in many species including the rat. In this study, using in silico and molecular biology techniques, we report the identification, cloning and characterization of rat Lyzl4 gene and also determine the expression pattern of Lyzl1, Lyzl3 and Lyzl6. The rat Lyzl genes were found to be distributed on three chromosomes and all of them retained the characteristic eight cysteine signature of c-type lysozyme. Homology modeling of rat LYZL4 indicated that its structure is similar to that of the mouse SLLP1. In the male reproductive tract of rat, Lyzl gene expression was confined to the testis. Lyzl1 and Lyzl4 were found to be expressed in tissues beyond the male reproductive tract, whereas Lyzl3 and Lyzl6 were not. Lyzl expression in the developing (10-60 day old) rats was androgen dependent in the testis. Immunodetection using antibodies against rat LYZL4 revealed the presence of LYZL4 protein in the germinal layer of the testes and on the sperm tail. Recombinant LYZL4 did not exhibit antibacterial, muramidase and isopeptidase activities characteristic to c-type lysozyme. To the best of our knowledge, for the first time we report the characterization of Lyzl genes in the rat. Results of our study indicate that rat LYZL proteins may have an important role in male reproductive tract function.     

Lyzl4, a novel mouse sperm-related protein, is involved in fertilization

The role of Chicken-type (c-type) lysozyme, a prototype lysozyme, in immunity has been characterized in many organisms. In this study, we cloned a novel c-type lysozyme-like gene, Lyzl4, which was located on mouse chromosome 9F4 and encoded 145 amino acids with a putative signal peptide and a protease cleavage site. The mature recombinant Lyzl4 protein expressed in yeast did not show the bacteriolytic activity. Sequence alignment analysis demonstrated that 3 of the 20 invariant residues in c-type lysozymes were changed in Lyzl4. One of the 'changed' amino acids (D52G) is located in the catalytic domain. Lyzl4 mRNA was selectively expressed in testis and epididymis in adult mice, with varying expression level across different developmental stages. High level of Lyzl4 protein was found on the spermatozoa of acrosomal region and principal piece of tail. Immuno-neutralization of Lyzl4 protein in spermatozoa with its specific antibody significantly decreased in vitro fertilization percentage in a dose-dependent manner, suggesting that Lyzl4 might be important for fertilization.     

SLLP1, a unique,intra-acrosomal,non-bacteriolytic,c lysozyme-like protein of human spermatozoa

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.     

The gene of chlamysin, a marine invertebrate-type lysozyme, is organized similar to vertebrate but different from invertebrate chicken-type lysozyme genes

In a recent publication we reported the protein purification, characterization, and the gene isolation of a cDNA encoding the antibacterial cold-active lysozyme-like protein chlamysin from the marine bivalve Chlamys islandica. A 4.2 kb genomic chlamysin gene has now been amplified and sequence-analyzed. By comparison to the cDNA sequence and its translation product, the coding region was found separated in four exons of 38-252 bp. The introns range in size from 0.8 to 1.5 kb, and have traditional spliceosomal intron 5'-GT donor and 3'-AG acceptor sites for splicing. Two of the introns contain multiple copies of three sequence motifs not found repeated in other published genes. The over-all gene organization of chlamysin resembles chicken-type (c-type) lysozyme genes in vertebrates, but is different from the three-exon structure in invertebrate c-type lysozyme genes. A phylogenetic analysis of invertebrate-type (i-type) and c-type lysozyme proteins demonstrated a large evolutionary distance between the i-type and the c-type enzyme classes. Exons of the i-type genes are not equally organized according to their homolog protein domains.     

Identification and expression analysis of three c-type lysozymes in Oreochromis aureus

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.     

Cloning and expression analysis of c-type and g-type lysozymes in yellow catfish (<Emphasis Type="Italic">Pelteobagrus fulvidraco</Emphasis>)

Lysozymes have important roles in innate immune system. Here, a c-type and a g-type lysozyme were identified from yellow catfish (Pelteobagrus fulvidraco). The deduced amino acid sequences of both lysozymes were conserved in catalytic sites and structural features as compared to their counterparts from other species. It was interesting that the g-type lysozyme possessed a signal peptide. The c-type and g-type lysozymes had the highest identity 89.4 and 76.2 % with that from channel catfish respectively. Phylogenetic analysis showed that the two lysozymes had a closely relationship with that from channel catfish and Astyanax mexicanus. Lysozymes from one order could form more than one clade in the phylogenetic tree, which indicated the gene duplications in evolution. Expression analysis with real time quantitative PCR revealed that the two lysozyme genes were constitutively expressed in all the tested tissues. The highest expression of c-type lysozyme was observed in liver, followed by spleen, head kidney, and trunk kidney, while the g-type lysozyme had highest expression in intestine, followed by spleen, head kidney, and trunk kidney. The mRNA levels of both genes were all up-regulated after challenging with Aeromonas hydrophila. However, there were differences in tissues and time points when the mRNA levels reached its peak between the two lysozymes. It indicated the diversity in regulation mechanisms and detailed functions among lysozymes. Taking together, these results will benefit the understanding of yellow catfish lysozymes.     

异育银鲫c型溶菌酶全长cDNA序列的生物信息学分析及其组织表达分析

利用RACE及克隆等方法获得了异育银鲫（Carassius auratus gibelio）c型溶菌酶基因全长cDNA序列．序列分析表明,所克隆的异育银鲫溶菌酶的cDNA全长751 bp,包括溶菌酶基因开放阅读框（ORF）438 bp,5′ 非编码区（UTR）为109 bp和3′ UTR为204 bp．438 bp ORF共编码146个氨基酸,其成熟肽的分子量预测值为14　543.6,理论等电点为8.86．通过ClustalW软件,将异育银鲫和其它多个物种c型溶菌酶的氨基酸序列进行多序列比对发现,所克隆的异育银鲫溶菌酶编码的氨基酸序列中存在c型溶菌酶的活性中心（Glu53和Asp69）,且与活性位点相邻的氨基酸序列高度保守．同时,8个保守的半胱氨酸残基也与其它物种的c型溶菌酶相一致．结合BLASTN分析的结果,可以确认所获得的异育银鲫溶菌酶cDNA序列属于c型溶菌酶．异育银鲫c型溶菌酶和人c型溶菌酶（pdb 1at6_）在蛋白质序列上有50%相似性,其三维（3-D）结构非常类似．通过氨基酸空间位置比较发现,两者具有类似的酶活中心,异育银鲫c型溶菌酶只能形成3个二硫键,比人少1个．荧光定量RT-PCR检测和溶菌酶活性测定显示,异育银鲫头肾和脾脏c型溶菌酶mRNA的表达量约为肝胰脏的2.9 倍和1.7 倍,异育银鲫头肾和脾脏的溶菌酶活性约为肝胰脏的6.2 倍和4倍．     

Lysozymes in the animal kingdom

Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme types have been identified — the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

The primary structure of donkey (Equus asinus) lysozyme contains the Ca(II) binding site of alpha-lactalbumin

The complete primary structure of donkey lysozyme has been established by pulsed liquid-phase sequencing of tryptic and chymotryptic peptides isolated by RP-HPLC. The positions of the Cys residues were identified by labeling the Cys residues with DABIA-reagent. Donkey lysozyme is a c-type lysozyme which is 129 amino acids long. It exhibits 50% homology to the human protein. We observe the full Ca(II) binding site suggested for the homologous alpha-lactalbumines. Although horse lysozyme has been reported to contain asparagine in position 61, which was in conflict with the three-dimensional structure of lysozyme, all other known c-type lysozymes, including donkey, contain Ser 61.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

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.     

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.     

Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity

Calcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and alpha-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows the most uncooperative refolding kinetics. Its partially-folded states serve as precursors for calcium-dependent self-assembly into ring-shaped and linear amyloids. The innate amyloid cytotoxicity of the ubiquitous lysozyme highlights the universality of this phenomenon and necessitates stringent measures for its prevention.