gallinacin-2-protein--chicken and gallinacin-1-protein--chicken

The aim of this study was to identify the types of gallinacin genes (GALs) expressed in ovarian follicles and to determine the changes in their expression during follicular growth and in response to lipopolysaccharide (LPS). Follicles at different stages of growth were collected from laying hens (n = 5) and LPS-injected hens (n = 3). The expression of GALs in the theca and granulosa layers was examined by semi-quantitative RT-PCR. The expression of GAL-1, -2, -7, -8, -10, and -12 in the theca layer and GAL-1, - 8, -10, and -12 in the granulosa layer was identified in white and yellow follicles. The expression of these genes was not changed in the theca and granulosa layers during follicular growth except for a decrease in that of GAL-1 in theca. The expression of GAL-1, -7, and -12 in the theca layer of the third largest follicles was increased in response to LPS at a dose of 1 mg/kg body weight and this increase was induced within 3 h and maintained until 12h postinjection. Granulosa layers did not respond to LPS until 12h injection. These results show that six and four types of GALs are expressed in the theca and granulosa layers of healthy follicles respectively, and their levels do not change with follicular growth except for GAL-1 in theca. Elevated levels of GAL-1, -7, and -12 expression in theca in response to LPS suggest that the theca cells expressing these GALs function to eliminate LPS-containing bacteria.

Topics: Animals; Avian Proteins; beta-Defensins; Chickens; Defensins; DNA Primers; Female; Gene Expression Regulation; Granulosa Cells; Lipopolysaccharides; Ovarian Follicle; Oviposition; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Theca Cells

Gallinacins (Gal) are antimicrobial peptides that play significant roles in innate immunity in chickens. The aim of this study was to examine whether age of birds and egg-laying activity (laying and non-laying caused by feed-regulation) affect the mRNA expression of Gal-1, -2, and -3 in the vagina of hens, and whether their expressions are changed in response to the stimulation with Salmonella enteritidis (SE) and lipopolysaccharide (LPS). White Leghorn hens were divided into groups of young and old laying hens, and groups of laying and non-laying hens after feed-regulation. Vaginal cells were cultured and stimulated with SE or LPS. Expressions of Gal-1, -2, and -3 mRNA in their vaginal mucosa and cultured cells were examined by quantitative real-time RT-PCR. The expressions of Gal-1, -2, and -3 of the vaginal mucosa were significantly greater in old birds than in young birds. Expression of these Gals in the vagina were decreased in the regressed oviduct of non-laying birds compared with laying birds. The expressions of Gal-1, -2, and -3 in the cultured vaginal cells were increased by stimulation with SE or LPS within 24 h. These results suggest that the mRNA expressions of Gal-1, -2 and -3 in the vagina of laying hens increased with age, whereas they decreased in the regressed oviduct during the non-laying phase. Also, synthesis of these antimicrobial peptides in the vagina may increase in response to SE and LPS to eliminate SE bacteria.

Topics: Animals; Avian Proteins; Base Sequence; Chickens; Defensins; DNA, Complementary; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Lipopolysaccharides; Molecular Sequence Data; Oviducts; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salmonella; Salmonella Infections, Animal; Time Factors; Transcription, Genetic; Vagina

Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, alpha-, beta-, and theta-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.. Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different beta-defensins but with no other groups of defensins being discovered. These chicken beta-defensin genes, designated as Gallinacin 1-13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 beta-defensin genes can be divided into two subgroups with Gallinacin 1-7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single beta-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution.. We conclude that the chicken genome encodes only beta-defensin sequences and that all mammalian defensins are evolved from a common beta-defensin-like ancestor. The alpha-defensins arose from beta-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and theta-defensins have arisen from alpha-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Avian Proteins; Bone Marrow; Chickens; Chromosome Mapping; Defensins; Evolution, Molecular; Expressed Sequence Tags; Gene Duplication; Genome; Humans; In Situ Hybridization, Fluorescence; Liver; Mammals; Mice; Molecular Sequence Data; Multigene Family; Organ Specificity; Phylogeny; Protein Isoforms; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Urogenital System

Four avian beta-defension prepropeptide cDNA sequences [gallinacins: Gal 1 (synonym CHP 1, chicken heterophil peptide 1), and Gal 2; turkey heterophil peptides: THP 1 and THP 2] were amplified from chicken or turkey bone marrow mRNA samples, respectively. Partial chicken beta-defensin cDNA sequences were obtained using degenerate primers based on chicken peptide sequences (Gal 1/CHP 1 and Gal 2). The complete cDNA sequences of the chicken beta-defensins were then determined by designing specific intrapeptidal primers, from the newly acquired sequence, and pairing one primer with a specific poly A primer tail sequence (3' end) and the other primer with an adapter primer in a 5' rapid amplification of cDNA ends (RACE) reaction. The two, turkey beta-defensins were amplified from turkey marrow using primers designed from chicken beta-defensin preproregions. The complete amino acid sequences for the prepropeptides were deduced for all four avian beta-defensins. Previously, only partial mature peptide sequences for the turkey beta-defensins and complete mature peptide sequences for the chicken beta-defensins were known. All sequences obtained translated accurately to complete and partial amino acid sequences reported for beta-defensins purified from chicken and turkey heterophil granules except for one additional amino acid for Gal 1/CHP 1. The four deduced beta-defensin proregions lack the long, negatively charged propiece reported in classical defensin proregions. These regions are thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule. Instead, these beta-defensin proregions are shorter and similar to storage granule-free beta-defensins proregions reported for bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). These are the first prepropeptide beta-defensins from leukocyte granules to be completely characterized.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Avian Proteins; Base Sequence; Bone Marrow; Cattle; Chickens; Defensins; Molecular Sequence Data; Peptides; Protein Precursors; RNA, Messenger; Turkeys

We purified three homologous antimicrobial peptides ('gallinacins') from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on-line LC-ESI-MS analysis of endo- and exoprotease peptide digests. The peptides contained 36-39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated beta-defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that beta-defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine beta-defensins are likely to constitute the essential primary structural motif of this ancient family of host-defense peptides.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Avian Proteins; Blood Bactericidal Activity; Blood Proteins; Chickens; Chromatography, High Pressure Liquid; Cysteine; Defensins; Leukocytes; Molecular Sequence Data; Peptides; Sequence Alignment; Sequence Homology, Amino Acid