Three-dimensional NMR structure of Hen Egg Gallin (Chicken Ovodefensin) reveals a new variation of the ß-defensin fold.

Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial ß-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel ß-sheet and the disulfide bridge array typical of vertebrate ß-defensins. Gallin can therefore be unambiguously classified as a ß-defensin. However, an additional short two-stranded ß-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of ß-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate ß-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.

Ovalbumin-related protein X is a heparin-binding ov-serpin exhibiting antimicrobial activities.

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys(367)-His(368) is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

Transcriptomic profiling of proteases and antiproteases in the liver of sexually mature hens in relation to vitellogenesis.

BACKGROUND: Most egg yolk precursors are synthesized by the liver, secreted into the blood and transferred into oocytes, to provide nutrients and bioactive molecules for the avian embryo. Three hundred and sixteen distinct proteins have been identified in egg yolk. These include 37 proteases and antiproteases, which are likely to play a role in the formation of the yolk (vitellogenesis), as regulators of protein metabolism. We used a transcriptomic approach to define the protease and antiprotease genes specifically expressed in the hen liver in relation to vitellogenesis by comparing sexually mature and pre-laying chickens showing different steroid milieu. RESULTS: Using a 20 K chicken oligoarray, a total of 582 genes were shown to be over-expressed in the liver of sexually mature hens (1.2 to 67 fold-differences). Eight of the top ten over-expressed genes are known components of the egg yolk or perivitelline membrane. This list of 582 genes contains 12 proteases and 3 antiproteases. We found that "uncharacterized protein LOC419301/similar to porin" (GeneID:419301), an antiprotease and "cathepsin E-A-like/similar to nothepsin" (GeneID:417848), a protease, were the only over-expressed candidates (21-fold and 35-fold difference, respectively) that are present in the egg yolk. Additionally, we showed the 4-fold over-expression of "ovochymase-2/similar to oviductin" (GeneID:769290), a vitelline membrane-specific protease. CONCLUSIONS: Our approach revealed that three proteases and antiproteases are likely to participate in the formation of the yolk. The role of the other 12 proteases and antiproteases which are over-expressed in our model remains unclear. At least 1/3 of proteases and antiproteases identified in egg yolk and vitelline membrane proteomes are expressed similarly in the liver regardless of the maturity of hens, and have been initially identified as regulators of haemostasis and inflammatory events. The lack of effect of sex steroids on these genes expressed in the liver but the products of which are found in the yolk suggests that these may be passively incorporated into the yolk rather than actively produced for that purpose. These results raise the question of the biological significance of egg yolk proteases and antiproteases, and more generally of all minor proteins that have been identified in egg yolk.

Antimicrobial potential of egg yolk ovoinhibitor, a multidomain Kazal-like inhibitor of chicken egg.

Chicken egg ovoinhibitor is a multidomain Kazal-type serine protease inhibitor with unknown function. Comparison of expression between different tissues indicated that ovoinhibitor is highly expressed in the magnum and liver followed by the uterus, which secrete egg white, egg yolk, and eggshell precursors, respectively. The results also revealed that ovoinhibitor expression is increased in the liver during sexual maturation followed by a subsequent decrease in mature hens. Ovoinhibitor was purified from the egg yolk plasma from nonfertilized eggs using two consecutive affinity chromatographies and gel filtration. Purified egg yolk ovoinhibitor was shown to inhibit trypsin and subtilisin. It was shown that purified egg yolk ovoinhibitor exhibited antimicrobial activities against Bacillus thuringiensis . The results suggest that this anti-protease plays a significant role in antibacterial egg defense against Bacillus spp., preventing contamination of table eggs (nonfertilized eggs) and protecting the chick embryo (fertilized eggs).

Cloning, expression and functional characterization of chicken CCR6 and its ligand CCL20.

Chemokines are key molecules that drive migration of lymphoid and myeloid cells toward organs in basal as well as inflammatory conditions. By recruiting immature dendritic cells to the mucosal surfaces, CCL20 acts in the very early events leading to the development of a specific immune response. In order to characterize dendritic cells in birds and better understand their role in the initiation of immune responses against pathogens of economic as well as human health relevance, we have cloned and expressed chicken CCL20 (chCCL20) and its specific receptor chCCR6. chCCL20 has 51% identity (60% similarity) with human CCL20, while the chicken receptor and its human counterpart display nearly 55% identity (and up to 70% similarity). chCCL20 and its specific receptor chCCR6 mRNAs are mainly expressed in bone marrow, secondary lymphoid organs and in the mucosal surfaces, in particular lungs and intestine. Both receptor and chemokine are functionally active when expressed as genuine or tagged proteins in mammalian expression systems, that is chCCR6 is mainly located at the cell surface within lipid rafts like its human counterpart. And secondly, both human and chicken chemokines were able to drive the migration of either chicken or human CCR6-transfected cells.