Natural antimicrobial peptides are present in different compartments (eggshell, egg white, and vitelline membranes) of the hen egg and are expected to be involved in the protection of the embryo during its development and to contribute to the production of pathogen-free eggs. In the present study, we used vitelline membranes from hen (Gallus gallus) eggs as a source of avian -defensin 11 (AvBD11). A purification scheme using affinity chromatography and reverse-phase chromatography was developed. Purified AvBD11 was analyzed by a combination of mass spectrometry approaches to characterize its primary sequence and structure. A monoisotopic molecular species at [M ؉ H]؉ of 9,271.56 Da was obtained, and its N-and C-terminal sequences were determined. We also examined posttranslational modifications and identified the presence of 6 internal disulfide bonds. AvBD11 was found to exhibit antimicrobial activity toward both Gram-positive and Gram-negative bacteria.The avian egg is a unique and original biological system which is formed according to a well-defined spatial and temporal sequence as it passes along the reproductive tract of the hen. The segments of the oviduct involved in the egg formation are the infundibulum, the magnum, the isthmus, and the uterus. Each segment expresses and secretes specific molecules that become successively incorporated into the vitelline membranes in the infundibulum, the egg white in the magnum, the eggshell membranes in the isthmus, and the eggshell in the uterus. Thus, the hen deposits into the egg, which can be considered a closed chamber, all the nutrients and protective systems that are necessary to support the development of an embryo during 21 days of incubation. Recently, transcriptomic and proteomic approaches have identified almost 1,000 putative proteins and peptides in the various compartments of eggs (6). Among these are a number of polypeptides that are likely to resist microbial contamination of the eggs, some of which belong to the family of avian -defensins (AvBDs).A total of 14 avian -defensin genes have been identified through in silico studies. Because most of them were simultaneously described by two research groups, some confusion was generated due to different nomenclatures (9,10,18,31). In a collaborative venture, we proposed a novel nomenclature that adopted the numbering system used by Xiao et al. (31) and replaced the term "gallinacin," used formerly by Lehrer's group, with "avian -defensin" (AvBD) (19). AvBDs are small cationic nonglycosylated peptides (1 to 9 kDa). Some of them were successfully tested for their antimicrobial activity, including chicken AvBDs AvBD1, -2, -7, and -9, turkey AvBD1 and -2, ostrich AvBDs AvBD1, -2, -7, and -8, and penguin AvBD103b (3, 30). In mammals, -defensins are molecules of innate and adaptive immunity, with a broad spectrum of antimicrobial activity (1). -Defensin molecules possess six highly conserved cysteines with the following consensus sequence motif, where C is a cysteine, G a glycine, and X any amino acid: X...
Three biologically active -defensins were purified by chromatography from chicken bone marrow extract: avian -defensin 1 (AvBD1), AvBD2, and the newly isolated -defensin AvBD7. Mass spectrometry analyses showed that bone marrow-derived AvBD1, -2, and -7 peptides were present as mature peptides and revealed posttranslational modifications for AvBD1 and AvBD7 in comparison to their in silico-predicted amino acid sequences. Tandem mass spectrometry analysis using the nanoelectrospray-quadrupole time of flight method showed N-terminal glutaminyl cyclization of mature AvBD7 and C-terminal amidation of mature AvBD1 peptide, while posttranslational modifications were absent in bone marrow-derived mature AvBD2 peptide. Furthermore, mass spectrometry analysis performed on intact cells confirmed the presence of these three peptides in mature heterophils. In addition, the antibacterial activities of the three -defensins against a large panel of gram-positive and -negative bacteria were assessed. While the three defensins displayed similar antibacterial spectra of activity against gram-positive strains, AvBD1 and AvBD7 exhibited the strongest activity against gram-negative strains in comparison to AvBD2.
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