Evolution of the bovine lysozyme gene family: Changes in gene expression and reversion of function

Recruitment of lysozyme to a digestive function in ruminant artiodactyls is associated with amplification of the gene. At least four of the approximately ten genes are expressed in the stomach, and several are expressed in nonstomach tissues. Characterization of additional lysozymelike sequences in the bovine genome has identified most, if not all, of the members of this gene family. There are at least six stomachlike lysozyme genes, two of which are pseudogenes. The stomach lysozyme pseudogenes show a pattern of concerted evolution similar to that of the functional stomach genes. At least four nonstomach lysozyme genes exist. The nonstomach lysozyme genes are not monophyletic. A gene encoding a tracheal lysozyme was isolated, and the stomach lysozyme of advanced ruminants was found to be more closely related to the tracheal lysozyme than to the stomach lysozyme of the camel or other nonstomach lysozyme genes of ruminants. The tracheal lysozyme shares with stomach lysozymes of advanced ruminants the deletion of amino acid 103, and several other adaptive sequence characteristics of stomach lysozymes. I suggest here that tracheal lysozyme has reverted from a functional stomach lysozyme. Tracheal lysozyme then represents a second instance of a change in lysozyme gene expression and function within ruminants. Correspondence to: D.M. Irwin     

Multiple cDNA sequences and the evolution of bovine stomach lysozyme

To investigate the origin of stomach expression of lysozyme in ruminants; we surveyed clones from a cow stomach cDNA library with a lysozyme cDNA probe. Ten percent of the clones in this library were lysozyme-specific. Thirty of the lysozyme clones were sequenced, and seven types of lysozyme mRNA sequence were found. They encode the three previously identified stomach isozymes of lysozyme. The seven sequences are closely related to one another and represent the products of a minimum of 4 of the approximately 10 cow lysozyme genes detected by genomic blotting. The most abundant form of stomach lysozyme (form 2) is encoded by at least two genes, whereas forms 1 and 3 are possibly each encoded by only one gene. The number of genes encoding each isozyme appears to contribute the largest factor in the relative abundance of each isozyme. The multiple lysozyme genes expressed in the cow stomach are the result of gene duplications that occurred during ruminant evolution. The recruitment of lysozyme as a major enzyme in the stomach may thus have involved an early regulatory event and a later 4-7-fold increase in expression allowed by gene amplification. During this period, the amino acid sequences of these lysozymes have been evolving more slowly than those of nonruminant lysozymes.     

Mosaic evolution of ruminant stomach lysozyme genes

The genomes of ruminant artiodactyls, such as cow and sheep, have approximately 10 lysozyme genes, 4 of which are expressed in the stomach. Most of the duplications of the lysozyme genes occurred 40-50 million years ago, before the divergence of cow and sheep. Despite this, the coding regions of stomach lysozyme genes within a species (e.g., cow, sheep, or deer) are more similar to each other than to lysozyme genes in other ruminants. This observation suggests that the coding regions of the stomach lysozyme genes have evolved in a concerted fashion. Our previous characterization of 3 cow stomach lysozyme genes suggested that it was only the coding exons that had participated in concerted evolution. To determine whether the introns and flanking regions of ruminant stomach lysozyme genes are evolving in a concerted or a divergent fashion, we have isolated and characterized 2 sheep stomach lysozyme genes. Comparison of the sequences of the sheep and cow stomach lysozyme genes clearly shows that the introns and flanking regions have evolved, like the 3' untranslated region of the mRNAs, in a divergent manner. Thus, if the four coding exons are evolving by concerted evolution, then a mosaic pattern of concerted and divergent evolution is occurring in these genes. The independent concerted evolution of coding exons of the ruminant stomach lysozyme gene may have assisted in the accelerated adaptive evolution of the lysozyme to new function in the early ruminant.     

Evolutionary genetics of ruminant lysozymes

Comparative studies of mammalian lysozymes and their genes have contributed to knowledge of how new functions arise during evolution. The recruitment of lysozymes for functioning in the stomach fluid of ruminants has occurred in response to selection pressures that are partly known and on a time-scale that is known. A semiquantitative analysis of adaptive evolution is thus made possible by the ruminant lysozyme system. Large-scale production of lysozyme by the stomach lining entailed gene duplication as well as a change in gene expression. Remoulding of the lysozyme for working and lasting in the stomach fluid involved accelerated amino acid replacements, which may have been facilitated by intergenic recombination. The possibility that multigene families can accelerate adaptive evolution, by virtue of their capacity for bringing together functionally coupled substitutions, receives emphasis in this review.     

Concerted evolution of ruminant stomach lysozymes. Characterization of lysozyme cDNA clones from sheep and deer

Contradictory evolutionary histories of ruminant lysozymes have been predicted by analysis of genomic blots (Irwin, D.M., Sidow, A., White, R., and Wilson, A.C. (1989) in The Immune Response to Structurally Defined Proteins: The Lysozyme Model (Smith-Gill, S.J., and Sercarz, E.E., eds) pp. 73-85, Adenine Press, Guilderland, NY) and sequences of cow stomach lysozyme cDNAs (Irwin, D.M., and Wilson, A.C. (1989) J. Biol. Chem. 264, 11387-11393). Genomic blots indicate that the amplification of the lysozyme gene family occurred 40-50 million years ago, while the cDNA sequences imply that the stomach genes began diverging from one another after the splitting of the deer and cow lineages, 25 million years ago. To resolve this contradiction, we characterized 111 stomach lysozyme cDNAs from two additional ruminant species: domestic sheep and axis deer. The cDNA sequences of the coding region of mature lysozyme together with the 3'-untranslated region were obtained from abomasum (true stomach) mRNA with the use of the polymerase chain reaction. The two primers for amplifying the cDNA were a lysozyme-specific primer, encoding a conserved sequence at the amino terminus of mature stomach lysozyme, and oligo(dT) as a general mRNA primer. Comparison of the cDNA sequences from these species to one another and to those of the cow revealed that different parts of the ruminant stomach lysozyme genes have had different evolutionary histories. The 3'-untranslated region has evolved in a divergent fashion since the original duplications 40-50 million years ago, supporting the genomic blot interpretation; by contrast, the coding region has evolved in a concerted fashion, that is, the multiple sequences within a species have evolved in unison. The 3'-untranslated portion of the lysozyme genes appears to have escaped from concerted evolution due to inability to initiate concerted evolution, rather than due to reduced sequence similarity. The process of concerted evolution in stomach lysozymes may have had roles both in adapting lysozyme to the stomach environment in early ruminants as well as in retarding amino acid sequence evolution in the well adapted lysozyme of modern ruminants.     

Stomach lysozyme gene of the langur monkey: Tests for convergence and positive selection

Summary Genomic blotting and enzymatic amplification show that the genome of the langur monkey (like that of other primates) contains only a single gene for lysozymec, in contrast to another group of foregut fermenters, the ruminants, which have a multigene family encoding this protein. Therefore, the langur stomach lysozyme gene has probably evolved recently (i.e., within the period of monkey evolution) from a conventional primate lysozyme. The sequences of cDNAs for the stomach lysozyme of langur and the conventional lysozymes of three other Old World monkeys were determined. Identification of the promoter for the stomach gene and comparison to the human gene, which is expressed conventionally in macrophages, show that both lysozyme genes use the same promoter. This suggests that the difference in expression patterns is due to change(s) in enhancer or silencer regulatory elements. With the cDNA sequences the hypothesis that the langur stomach lysozyme has converged in amino acid sequence upon the stomach lysozymes of ruminants is tested. Consistent with the convergence hypothesis, only those sites that specify amino acids in the mature lysozyme are shared uniquely with ruminant lysozyme genes. None of the silent sites at third positions of codons or in noncoding regions support a link between the langur and ruminants. Statistical analysis based on silent sites rules out the possibility of horizontal transfer of a stomach lysozyme gene between the langur and ruminant lineages and supports the close relationship of the langur lysozyme gene to that of other monkeys.     

Physical mapping of the lysozyme gene family in cattle

Amplification of an ancestral lysozyme gene in artiodactyls is associated with the evolution of foregut fermetation in the ruminant lineage and has resulted in about ten lysozyme genes in true ruminants. Hybridization of a cow stomach lysozyme 2 cDNA clone to restricted DNAs of a panel of cowxhamster hybrid cell lines revealed that all but one of the multiple bovine-specific bands segregate concordantly with the marker for bovine syntenic group U3 [Chromosome (Chr) 5]. The anomalous band was subsequently mapped to bovine syntenic group U22 (Chr 7) with a second panel of hybrids representing all 31 bovine syntenic groups. By two-dimensional pulsed-field gel electrophoresis the lysozyme genes on cattle Chr 5 were shown to be clustered on a 2- to 3-Mb DNA fragment, while the lactalbumin gene and pseudogenes that are paralogous and syntenic with the lysozymes were outside the lysozyme gene cluster. Chromosomal fluorescence in situ hybridization of a cocktail of lysozyme genomic clones localized the lysozyme gene cluster to cattle Chr 5 band 23, corroborating the somatic cell assignment.     

Amino acid sequences of stomach and nonstomach lysozymes of ruminants

Summary Complete amino acid sequences are presented for lysozymesc from camel and goat stomachs and compared to sequences of other lysozymesc. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants.     

Molecular genetics and evolution of stomach and nonstomach lysozymes in the hoatzin

Multiple genes of the hoatzin encoding stomach lysozyme c and closely related members of this calcium-binding lysozyme c group were cloned from a genomic DNA library and sequenced. There are a minimum of five genes represented among these sequences that encode two distinct groups of protein sequences. One group of three genes corresponds to the stomach lysozyme amino acid sequences, and the remaining genes encode predicted proteins that are more basic in character and share several sequence identities with the pigeon egg-white lysozyme rather than with the hoatzin stomach lysozymes. Despite these structural similarities between some of the hoatzin gene products and the pigeon lysozyme, phylogenetic analyses indicate that all of the hoatzin sequences are closely related to one another. This is borne out by the relatively small genetic distances even in the intronic regions, which are not subject to the selective pressures operating on the coding regions of the stomach lysozymes. These results suggest that multiple gene duplication events have occurred during the evolution of hoatzin lysozymes.     

Stomach lysozymes of ruminants. II. Amino acid sequence of cow lysozyme 2 and immunological comparisons with other lysozymes

The complete sequence of 129 amino acids has been determined for one of three closely related lysozymes c purified from cow stomach mucosa. The sequence differs from those known for 17 other lysozymes c at 39-60 positions, at one of which there has been a deletion of 1 amino acid. The glutamate replacement at position 101 and the deletion of proline at position 102 eliminate the aspartyl-prolyl bond that is present between these positions in all other mammalian lysozymes c tested. This bond appears to be the most acid-sensitive one in such lysozymes at physiological temperature. Of the 40 positions previously found to be invariant among lysozymes c, only one has undergone substitution in the cow lineage. This modest number of changes at novel positions is consistent with the inference, based on tree analysis and antigenic comparisons, that the tempo of evolutionary change in the cow lysozyme lineage has not been radically different from that in other lysozyme c lineages. The mutations responsible for the distinctive catalytic properties and stability of cow lysozyme c could be a minor fraction of the total that have been fixed in the cow lineage.     

Molecular adaptation of a leaf-eating bird: stomach lysozyme of the hoatzin

This report describes a lysozyme expressed at high levels in the stomach of the hoatzin, the only known foregut-fermenting bird. Evolutionary comparison places it among the calcium-binding lysozymes rather than among the conventional types. Conventional lysozymes were recruited as digestive enzymes twice in the evolution of mammalian foregut fermenters, and these independently recruited lysozymes share convergent structural changes attributed to selective pressures in the stomach. Biochemical convergence and parallel amino acid replacements are observed in the hoatzin stomach lysozyme even though it has a different genetic origin from the mammalian examples and has undergone more than 300 million years of independent evolution.      

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.     

Yak (Bos Grunniens) Stomach Lysozyme: Molecular Cloning,Expression and its Antibacterial Activities

The cDNA coding for stomach lysozyme in yak was cloned. The cloned cDNA contains a 432 bp open reading frame and encodes 143 amino acids (16.24 KDa) with a signal peptide of 18 amino acids. Further analysis revealed that its amino acid sequence shares many common properties with cow milk lysozyme. Expression of this gene was also detected in mammary gland tissue by RT-PCR. Phylogenetic relationships among yak stomach lysozyme and 8 cow lysozymes indicated that the yak enzyme is more closely related to both cow milk lysozyme and the pseudogene ΨNS4 than cow stomach lysozyme. Recombinant yak lysozyme purified by Ni²⁺-column showed a molecular weight of 33.78 kDa and exhibited lytic activity against Staphylococcus aureus, providing evidence of its antibacterial activities.     

Stomach lysozymes of the three-toed sloth (Bradypus variegatus), an arboreal folivore from the Neotropics

Lysozymes are antimicrobial defences that act as digestive enzymes when expressed in the stomach of herbivores with pre-gastric fermentation. We studied this enzyme in the complex stomach of the three-toed sloth (Bradypus variegatus), a folivore with pre-gastric fermentation. Lysozymes were identified by SDS-PAGE and immunoblotting in all portions: diverticulum, pouch, glandular and muscular prepyloric area with 14.3 kDa of molecular mass. Purified lysozymes from all areas but the diverticulum were characterized by MALDI-TOF, optimal pH, optimal ionic strength, and specific activity. The differences observed suggested at least three isoforms. The optimal pHs were similar to the pH of the stomach portion where the enzymes were isolated. The lysozyme from the pouch (fermentation chamber) exhibited higher specific activity and concentration than the others. The specific activity of the enzyme from the acid muscular prepyloric portion was comparable to that reported in the cow abomasums; however, its concentration was lower than that observed in cow. This distinctive pattern of secretion/specific activity and overall low concentration suggests different roles for the lysozymes in this herbivore compared to Artiodactyla. We postulate that sloth stomach lysozymes may still be antimicrobial defences by protecting the microbial flora of the fermentation chamber against foreign bacteria.     

日本鳗鲡的C-型和G-型溶菌酶研究

研究以日本鳗鲡(Anguilla japonica Temminck et Schlegel)为研究对象, 根据其基因组数据库, 预测并扩增出2类, 共5个溶菌酶基因, 包括1个C-型溶菌酶和4个G-型溶菌酶, 分别命名为AJLysC、AJLysG1、AJLysG2、AJLysG3和AJLysG4。它们的cDNA全长分别为811、749、1352、1175和733 bp, 编码143、193、185、185和187个氨基酸。SignalP预测表明, AJLysC和AJLysG1的N-端分别包括15和19氨基酸的信号肽, 另外3种溶菌酶没有信号肽。基因组分析显示, AJLysC、AJLysG2、AJLysG3和AJLysG4的基因结构与其他鱼类的同类溶菌酶的基因结构相似, C-型溶菌酶具有4个外显子, G-型则具有5个。但是, AJLysG1的基因结构与其他鱼类G-型溶菌酶不同, 具有6个外显子, 与其他鱼类溶菌酶的蛋白序列比较, 发现AJLysG1缺失其他G-型溶菌酶存在的第2个酶活性位点氨基酸, 即天冬氨酸Asp。AJLysC与其他很多物种的C-型溶菌酶具有较高的同一性, 如与牙鲆的同一性为72.7%。G-型溶菌酶中AJLysG2、AJLysG3、AJLysG4彼此之间以及与其他物种G-型溶菌酶的同一性相对较高; 而AJLysG1与其他物种以及与其他3种G-型溶菌酶的同一性均不高, 且都在50%以下。组织表达分析显示, 所有5个溶菌酶基因在12种检测的组织中均有表达。C-型溶菌酶在胃及免疫相关组织的表达量较高; G-型溶菌酶在各组织/器官中的表达则差异较大, AJLysG1在皮肤和肌肉中的表达量最高, AJLysG2在免疫组织/器官如血液、头肾、体肾和鳃中表达量较高。经迟缓爱德华氏菌(Edwardsiella tarda)刺激48h后, 这5个溶菌酶基因在组织/器官中的表达量均有上调, 其中在血液、肠道和头肾等的上调较为显著。此外, 研究尝试重组表达这些抗菌肽, 获得了AJLysG2、AJLysG3和AJLysG4基因在鲤上皮瘤细胞(Epithelioma papulosum cyprinid, EPC)细胞中的表达, 重组蛋白表现出对溶壁微球菌(Micrococcus lyso-deikticus)生长的明显抑制作用。文章较全面地研究了日本鳗鲡溶菌酶基因的组成和类型及其表达变化, 并重组表达了部分基因, 这为进一步研究这些溶菌酶的功能, 特别是对病原微生物的作用奠定了基础。     

The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract

Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles in immunity and digestion, we have now characterized all six lysozyme genes in the cloned part of the lysozyme locus at 61F, and a seventh gene that maps to the same chromosomal location. The expression of the genes follows four different patterns: firstly, four closely related genes, LysB, C, D and E, are all strongly expressed in the midgut of larvae and adults; secondly, LysP is expressed in the adult salivary gland; thirdly, LysS is expressed mainly in the gastric caecae of larvae; and finally, LysX is primarily expressed in the metamorphosing midgut of late larvae and early pupae. The LysD-like genes and LysS are strongly repressed in artificially infected animals, possibly reflecting a malaise reaction in the digestive tract. None of the genes is expressed in the fat body or haemocytes. Thus rather than being a component of the haemolymph, the Drosophila lysozymes are found mainly in the digestive tract where they are expressed at a high level. Furthermore all genes, except LysP, encode acidic proteins, in contrast to the strongly basic “typical” lysozymes. This is highly reminiscent of the situation in ruminants, where the lysozymes have been recruited for the digestion of symbiotic bacteria in the stomach.     

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.     

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)     

The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract

Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles in immunity and digestion, we have now characterized all six lysozyme genes in the cloned part of the lysozyme locus at 61F, and a seventh gene that maps to the same chromosomal location. The expression of the genes follows four different patterns: firstly, four closely related genes, LysB, C, D and E, are all strongly expressed in the midgut of larvae and adults; secondly, LysP is expressed in the adult salivary gland; thirdly, LysS is expressed mainly in the gastric caecae of larvae; and finally, LysX is primarily expressed in the metamorphosing midgut of late larvae and early pupae. The LysD-like genes and LysS are strongly repressed in artificially infected animals, possibly reflecting a malaise reaction in the digestive tract. None of the genes is expressed in the fat body or haemocytes. Thus rather than being a component of the haemolymph, the Drosophila lysozymes are found mainly in the digestive tract where they are expressed at a high level. Furthermore all genes, except LysP, encode acidic proteins, in contrast to the strongly basic typical lysozymes. This is highly reminiscent of the situation in ruminants, where the lysozymes have been recruited for the digestion of symbiotic bacteria in the stomach.     

Characterization of the cow stomach lysozyme genes: Repetitive DNA and concerted evolution

Cow stomach lysozyme genes have evolved in a mosaic pattern. The majority of the intronic and flanking sequences show an amount of sequence difference consistent with divergent evolution since duplication of the genes 40–50 million years ago. In contrast, exons 1, 2, and 4 and immediately adjacent intronic sequences differ little between genes and show evidence of recent concerted evolution. Exon 3 appears to be evolving divergently. The three characterized genes vary from 5.6 to 7.9 kilobases in length. Different distributions of repetitive DNA are found in each gene, which accounts for the majority of length differences between genes. The different distributions of repetitive DNA in each gene suggest the repetitive elements were inserted into each gene after the duplications that give rise to these three genes and provide additional support for divergent evolution for the majority of each gene. The observation that intronic and flanking sequences are evolving divergently suggests that the concerted evolution events involved in homogenizing the coding regions of lysozyme genes involve only one exon at a time. This model of concerted evolution would allow the shuffling of exon-sized pieces of information between genes, a phenomenon that may have aided in the early adaptive evolution of stomach lysozyme.Deceased July 21, 1991 Correspondence to: D.M. Irwin