The chicken egg possesses physical and chemical barriers to protect the embryo from pathogens. OCX-36 (ovocalyxin-36) was suggested to be a 36 kDa eggshell-specific protein that is secreted by the regions of the oviduct responsible for eggshell formation. Its expression is strongly up-regulated during shell calcification. This protein was also detected in vitelline membrane and expressed in gut tissues. Analysis of the OCX-36 protein sequence revealed that OCX-36 is related to the BPI (bactericidal permeability-increasing proteins)/LBP [LPS (lipopolysaccharide)-binding proteins]/PLUNC (palate, lung and nasal epithelium clone) superfamily, and that there are strong similarities between the exon/intron organization of the mammalian LBP/BPI and the avian OCX-36 genes. A recent study revealed that OCX-36 originates from a tandem duplication of an ancestral BPI/LBP/PLUNC gene, after the divergence of birds and mammals. Its antimicrobial activity was recently investigated and it was shown that OCX-36 binds to LPS from Escherichia coli. High-throughput methodologies have led to the identification of approximately 1000 new egg proteins. Among these are LBP/BPI proteins that might play a role in the natural defences of the egg to protect the embryo during its development in the external milieu, and may function to keep the table egg free of pathogens. The function of these BPI-like molecules is the subject of intense research to characterize their putative LPS-binding properties and antimicrobial activity.

Introduction

The chicken egg is formed in the hen's left ovary and oviduct. The ovary supports the accumulation of egg-yolk proteins and maturation of the ovum. After ovulation, the yolk enters the oviduct, where albumen, eggshell membranes and the eggshell are sequentially deposited in the different segments of the hen reproductive tract (magnum, white isthmus and uterus respectively). The original role of the chicken egg is to be a completely self-sufficient and aseptic biological package for the extra-uterine development of the embryo. This role as a natural container means that the egg must contain all the components (vitamin, proteins, lipids and minerals) that are essential for the development from a reproductive cell to a viable chick in 21 days. To ensure this function, the egg possesses a broad range of proteins with various biological activities, and many of them are involved in natural defences [1,2]. The egg is also a basic nutrient for human consumption and has a high nutritive value and is a well-balanced source of amino acids that are easily assimilated [14]. Moreover, the chicken egg is a major source of active molecules such as anti-hypertensive, anti-cancer, anti-oxidant, cryoprotective, immunomodulating and anti-adhesive components [1,2,5].

The egg also contains protective systems to resist physical and/or microbial assaults. The first natural defence of the egg is the eggshell, which acts as a physical barrier against bacteria as long as it remains intact [6,7]. The eggshell is a highly ordered structure made of calcium carbonate and an organic matrix. The chicken eggshell matrix is a complex mixture of proteins and polysaccharides [810] that plays a crucial role in the control of mineralization and in determining the mechanical properties of the shell [6,1113]. The second natural defence of the egg is a chemical protection system consisting of yolk, egg white and eggshell proteins with antimicrobial properties [1,2,5,14].

In this review, we report the presence of OCX-36 (ovocalyxin-36), a chicken egg protein related to the BPI (bactericidal permeability-increasing proteins)/LBP [LPS (lipopolysaccharide)-binding protein]/PLUNC (palate, lung and nasal epithelium clone) superfamily. Moreover, the recent developments of high-throughput methods (proteomics and transcriptomics) have allowed the identification of several hundred new egg components. Among these are additional proteins with a BPI/LBP/PLUNC-like domain, which are reported in the present article.

OCX-36, a chicken eggshell protein related to LBP/BPI/PLUNC family proteins

The chicken eggshell is a sophisticated composite calcitic bioceramic, which possesses unique mechanical properties deriving from its complex polycrystalline structure. Eggshell mineralization occurs in the distal part of the oviduct (uterus), in an acellular milieu (uterine fluid), where the physical and chemical requirements necessary for biomineralization are present [11,13,15]. The eggshell is made of calcium carbonate in calcitic form, which is associated with an organic matrix composed of proteins, glycoproteins and proteoglycans. These components are thought to influence the fabric of this biomaterial [8,10,11,13]. OCX-36 is an eggshell matrix protein that corresponds to a 36 kDa electrophoretic band and is highly abundant in the uterine fluid during the active growth phase of calcification [16]. It was identified and characterized by a combination of biochemistry, molecular biology, bioinformatics and data mining procedures. A specific antibody against OCX-36 was produced and used to screen a chicken uterine cDNA expression library. Positives clones were sequenced and the consensus sequence was combined with ESTs (expressed sequence tags) to obtain a full-length cDNA sequence of 1995 bp, which corresponds to a translated mature protein of 438 amino acids and a predicted mass of 46.5 kDa. The cDNA and protein sequences were novel and it was therefore named ovocalyxin-36 (ovum for egg, calyx for shell and 36 for its apparent molecular mass by SDS/PAGE) [16]. Immunolocalization of OCX-36 by immunofluorescence and colloidal-gold immunocytochemistry has confirmed that OCX-36 is present throughout the entire eggshell, with the highest abundance in the eggshell membranes [16]. This localization in the eggshell was further confirmed by an eggshell proteomic survey that revealed an overall total of 528 different proteins as constituents of the eggshell matrix [17,18]. Additionally, OCX-36 was also detected in vitelline membranes using MS-based high-throughput methods for protein identification [19].

Both mRNA expression and protein synthesis take place in the chicken reproductive tract, more specifically in the tissue where eggshell calcification takes place (Figure 1a) [16]. OCX-36 protein was detected in white isthmus, red isthmus and uterus. Recently, OCX-36 mRNA was also found to be expressed in different segments of the gut [20]. Two types of uterine secretory cells, those of the surface epithelium and of the underlying tubular glands, are involved in secretion of eggshell matrix proteins. OCX-36 is secreted by the tubular gland cells [16]. Physiological regulation of OCX-36 gene expression during egg formation was investigated [16,21]. The expression of OCX-36 mRNA was basal in uterus from birds with an undeveloped oviduct prior to sexual maturity; expression was up-regulated by sexual maturity and the accompanying onset of egg production. Finally, OCX-36 uterine expression was greatest when an egg was present in the uterus during eggshell calcification; expression was 18-fold higher in uterus of hens sampled with a calcified egg undergoing eggshell deposition, compared with empty uterus when no shell was forming and the egg was in the proximal segment of the oviduct (Figure 1b) [16].

Expression of OCX-36 mRNA [16]

Figure 1
Expression of OCX-36 mRNA [16]

(a) Reverse transcription–PCR to detect OCX-36 mRNA in different parts of the oviduct (Ma, magnum; WI, white; RI, red isthmus; Ut, uterus) and from liver (Liv), kidney (Kid), duodenum (Duod) and muscle (Mus). Std, standards; W, water (control). (b) Real-time reverse transcription–PCR to quantify the levels of OCX-36 expression in uterus when the egg was in the magnum during the egg white deposition (3 h after ovulation) and when a hard-shelled egg was forming in uterus (18 h after ovulation). Modified from Gautron et al. [16] with permission.

Figure 1
Expression of OCX-36 mRNA [16]

(a) Reverse transcription–PCR to detect OCX-36 mRNA in different parts of the oviduct (Ma, magnum; WI, white; RI, red isthmus; Ut, uterus) and from liver (Liv), kidney (Kid), duodenum (Duod) and muscle (Mus). Std, standards; W, water (control). (b) Real-time reverse transcription–PCR to quantify the levels of OCX-36 expression in uterus when the egg was in the magnum during the egg white deposition (3 h after ovulation) and when a hard-shelled egg was forming in uterus (18 h after ovulation). Modified from Gautron et al. [16] with permission.

Analysis of the OCX-36 protein sequence [16] revealed significant alignments with mammalian LBP, BPI and CETP (cholesteryl ester-transport protein) and with PLUNC family proteins [2225]. The sequence comparison reveals that OCX-36 is related to the antibacterial protein superfamily BPI/LBP/PLUNC with 20–27% identities between OCX-36 and BPI/LBP/PLUNC members [BPIL3, LPLUNC3, LPLUNC4 and LPLUNC6) [16,20,26]. The similarity for conservative substitutions ranges from 39 to 44% and the Blast E-values between OCX-36 and these proteins indicated a significant relationship. OCX-36 homology with LBP and BPI proteins is confirmed by database searching for protein domains and functional sites. The region of similarity extends over the entire length of the OCX-36 sequence. Residues 33–244 and 273–439 of the OCX-36 amino acid sequence corresponded to the N-terminal and C-terminal domains of the LBP/BPI family proteins respectively. The genes coding for bactericidal/permeability increasing-like (BPI-like) proteins are organized in two loci that are syntenous across animal genomes. Most members are encoded by a region on chromosome 20 [27]. OCX-36 maps to GGA20, which presents a conservation of synteny with the portion of HSA20 containing LBP and BPI [16]. The OCX-36 gene is nested in a tandemly arranged BPI/LBP/PLUNC gene cluster on the chromosome 20, and it is in particular next to BPIL3, LPLUNC3, LPLUNC4 (Figure 2) [26] and LPLUNC6 [20]. Synteny analysis reveals that the BPI/LBP/PLUNC gene cluster is conserved in amniotes (including both birds and mammals), except that OCX-36 is specifically present in the chicken genome but not in any mammalian genomes. Homology between OCX-36 and the LBP/BPI family of mammalian proteins is further reinforced by comparison of their gene structure. The exon/intron organization of the OCX-36 gene [16] is very similar to that of the highly conserved LBP and BPI genes [28], with most corresponding exons possessing identical sizes (Figure 3). Recently, evolutionary scenarios of egg-specific genes were established to evaluate the significance of gene formation, gene divergence and gene loss that are associated with the avian egg [26]. OCX-36 protein has originated by specific gene formation in birds. OCX-36 gene arises from a tandem duplication of an ancestral BPI/LBP/PLUNC gene after the divergence of birds and mammals. Chicken and zebra finch OCX-36 exhibit a large degree of similarity throughout the protein sequence (56% identity), indicating great conservation for this matrix protein between these species that are separated by almost 100 million years of evolution [29].

Gene formation of the eggshell-expressed OCX-36 in G. gallus

Figure 2
Gene formation of the eggshell-expressed OCX-36 in G. gallus

(A) Consensus phylogenetic tree of OCX-36 and its relatives reconstructed by the fusion of four separate methods. Numbers on the right of pentagons are gene locations expressed in Mb for different species. NJ, neighbour joining; ME, minimum evolution; MP, maximum parsimony; ML, maximum likelihood. The branches with bootstrap values 80% calculated by at least three methods are shown in bold. (B) The genomic localization of OCX-36 in the G. gallus and syntenic comparison among four vertebrate species (Homo sapiens, Monodelphis domestica, G. gallus and Oryzias latipes). Numbers on the right of pentagons are gene locations expressed in Mb for different species. Reprinted from Tian et al. [26] with permission.

Figure 2
Gene formation of the eggshell-expressed OCX-36 in G. gallus

(A) Consensus phylogenetic tree of OCX-36 and its relatives reconstructed by the fusion of four separate methods. Numbers on the right of pentagons are gene locations expressed in Mb for different species. NJ, neighbour joining; ME, minimum evolution; MP, maximum parsimony; ML, maximum likelihood. The branches with bootstrap values 80% calculated by at least three methods are shown in bold. (B) The genomic localization of OCX-36 in the G. gallus and syntenic comparison among four vertebrate species (Homo sapiens, Monodelphis domestica, G. gallus and Oryzias latipes). Numbers on the right of pentagons are gene locations expressed in Mb for different species. Reprinted from Tian et al. [26] with permission.

Comparison of the intron/exon organization for human LBP, human BPI and chicken OCX-36 genes [16]

Figure 3
Comparison of the intron/exon organization for human LBP, human BPI and chicken OCX-36 genes [16]

Exons (Roman numerals) and length (Arabic numerals) are indicated. Conserved exon lengths are emphasized by black boxes. Introns are not drawn to scale. Human BPI, accession number NM_001725; human LBP, accession number NM_004139. Reprinted from Gautron et al. [16] with permission.

Figure 3
Comparison of the intron/exon organization for human LBP, human BPI and chicken OCX-36 genes [16]

Exons (Roman numerals) and length (Arabic numerals) are indicated. Conserved exon lengths are emphasized by black boxes. Introns are not drawn to scale. Human BPI, accession number NM_001725; human LBP, accession number NM_004139. Reprinted from Gautron et al. [16] with permission.

An antimicrobial role for OCX-36 protein has been proposed since its protein sequence shares significant similarity with LBP, BPI and PLUNC family proteins. These proteins are well known in mammals for their involvement in defence against bacteria. They belong to the superfamily of proteins known to be key components of the innate immune system, which act as the first line of host defence [23]. LBP binds the lipid A component of the LPS layer of Gram-negative bacteria and transfers them to CD14, an LPS receptor [30]. BPI also binds LPS, followed by permeabilization of the cytoplasmic membrane and a decrease in the electrochemical gradient of the bacterial cell leading to death [31]. An efficient extraction and purification method of OCX-36 protein from eggshell membrane has been recently developed [32]. The purified OCX-36 showed significant binding activity for LPS derived from Escherichia coli, and could modestly inhibit the bacterial growth of Bacillus subtilis, Staphylococcus aureus, E. coli and Pseudomonas aeuginosa [32]. These results are functional evidence suggesting that OCX-36 may play a role as an antimicrobial protein in the egg.

Additional LBP/BPI-like proteins in the chicken egg

During the last 10 years, the results of functional genomic studies have dramatically transformed biology and biotechnology, including egg science. The recent development of high-throughput methods used in combination with the newly available chicken genomic sequence [33] and the development of bioinformatic tools to predict functions have generated new insights for the characterization of new and minor egg components [34]. The recently assembled databases containing a large number of chicken gene transcripts and proteins sequences enabled the exploration of egg compartment proteomes, using MS-based high-throughput methods. Recently analysed proteomes are those of the organic matrix of the chicken calcified eggshell, egg white, egg yolk and vitelline membrane [1719,3538]. Moreover, the chicken oviduct offers significant advantages for a transcriptomic approach. Egg formation in the oviduct occurs daily by sequential secretion on to the yolk of the various compartments of the egg. Each segment of the oviduct: the magnum, isthmus and uterus, has a very specific role in the synthesis of constituents of various egg compartments. The entire oviduct originates from the same population of cells, which specialize at sexual maturity into specific regions responsible for the deposition of egg white (magnum), eggshell membranes (white isthmus) and calcified shell (uterus). Consequently, the comparison of gene expression in the various segments of the oviduct reveals the genes that encode proteins synthesized and secreted in each specialized region of the oviduct, and is another way to catalogue the constituents of each egg compartment. Two recent reports have analysed global gene expression in hens' reproductive tract [21,39]. These recent developments allowed the identification of hundreds of proteins potentially involved in various physiological processes associated with egg formation. The availability of software that allows large amounts of information contained in databases to be interrogated, either using data from the literature (text mining), or classifications using protein sequences in different ontologies or pathways allows a broader view of the functions and of the biological relevance of the detected proteins. Using these bioinformatic analyses, new candidate proteins that could have a major role in egg defence were identified [40]. Among these are three additional BPI/LBP-like proteins.

One of these, the TENP protein from Gallus gallus, was originally identified as a transiently expressed gene in neural precursor cells in retina and brain, and had been proposed to function in the transition to cell differentiation in neurogenesis. TENP was immunodetected in cellular membrane fractions, implying that it might be a membrane protein, as predicted by a computer analysis of its primary sequence [41]. More recent studies indicate that TENP protein is present in every chicken egg compartment (eggshell, egg white, vitelline membranes and egg yolk) [17,3538,42], and in emu egg white [43]. Little is known about TENP. It is notable that this protein was recently identified in a proteomic analysis of host proteins associated with purified coronavirus infectious bronchitis virus particles [44]. Both phylogeny and genomic comparison suggest that the chicken TENP gene is orthologous to the mammalian BPIL1 (Figure 2). Probably, we observe a case of functional divergence after duplication event between TENP and BPIL1 genes [26]. The BPIL2 protein was also detected in egg white and in vitelline membranes [37,38]. Uterine BPIL2 expression was found to be strongly up-regulated in sexually matured hens compared with juveniles with an undeveloped oviduct [21]. Finally, the last BPI/LBP-like protein identified in the egg is the ‘similar-to-BPI protein’ (XP_425484, LOC427911), which is detected in egg white [35].

Avian BPI-like genes are located on the corresponding chicken chromosome 20, where the OCX-36 gene is nested in the tandemly arranged BPI/LBP/PLUNC gene cluster; it is next to BPIL3, LPLUNC3, LPLUNC4 and LPLUNC6 [20,26]. TENP is also localized in the same locus in the chicken genome (Figure 2). Two other BPI-like proteins are encoded by a locus on chromosome 1 in chickens; BPI-Like-2 (LOC771461) and similar-to-BPI (LOC427911). The co-localization of these genes is conserved in mammalian genomes (shared synteny). Examination of the EST database for tissue-specific expression of BPI-like genes indicates that OCX-36, TENP, BPI-Like-2 (BPIL2) and similar-to-BPI are expressed in chicken reproductive tissues; moreover, sensitive proteomic scans of egg compartment proteins reveal that these members of the BPI-like family are present at detectable levels.

Conclusion

The development of molecular biology in the late 1980s, the recent publication of the chicken genome sequence and the development of new bioinformatic tools were major scientific advances leading to the identification and characterization of several egg components that had not been previously identified. The newly identified molecules have stimulated research to understand avian biology and identify biologically active compounds that might have potential interest for pharmaceutical, cosmetic and food industries. Analysis of the egg molecules showed the presence of BPI/LBP/PLUNC-like proteins, the function of which is the subject of intense research to characterize their putative LPS-binding properties and antimicrobial activity.

The biological activities of egg molecules that constitute the natural defences of the egg are represented by antimicrobial activities or those involved in eggshell formation; these can be reinforced by selection or by controlling the environmental factors affecting their level of activity. Some of the novel egg proteins are candidates to be used as biological markers for marker-assisted selection to reinforce the protective systems of the egg [45]. Alternatively, the study of environmental and breeding factors that might affect their activity or levels can be used to optimize egg defences. Together, these approaches will contribute to reduce the risk of food-borne disease outbreaks for egg consumers.

Proteins with a BPI/LBP/PLUNC-Like Domain: Revisiting the Old and Characterizing the New: A Biochemical Society Focused Meeting held at New Business School, University of Nottingham, U.K., 5–7 January 2011. Organized and Edited by Colin Bingle (Sheffield, U.K.) and Sven-Ulrik Gorr (University of Minnesota School of Dentistry, Minneapolis, MN, U.S.A.).

Abbreviations

     
  • BPI

    bactericidal permeability-increasing protein

  •  
  • EST

    EST, expressed sequence tag

  •  
  • LPS

    lipopolysaccharide

  •  
  • LBP

    LPS-binding protein

  •  
  • OCX-36

    ovocalyxin-36

  •  
  • PLUNC

    palate, lung and nasal epithelium clone

We acknowledge Xin Tian for her help with phylogenetic studies.

Funding

We gratefully acknowledge the European Community for its financial support to Egg Defence [grant number QLRT-2001-01606], RESCAPE [grant number RESCAPE Food CT 2006-036018] and SABRE (European Integrating Project Cutting-Edge Genomics for Sustairable Animal Breeding).

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