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Eggshell matrix proteins related to antibacterial proteins: J. Gautron et al. Chicken eggshell matrix proteins related to anti-bacterial protein families J. GAUTRON1*, E. MURAYAMA1, M.T. HINCKE 2 and Y. NYS 1 1 Station de recherches avicoles, INRA, 37380 Nouzilly, France Department of Cellular and molecular medicine, University of Ottawa, Ottawa K1H 8M5, Canada *[email protected] 2 Keywords: hen; eggshell; matrix proteins; anti-microbial, anti-bacterial; egg defences Summary Chicken eggshell matrix proteins have been largely investigated during the last decade. Proteins and associated genes has been identified and characterised. Experimental evidence suggests a role for matrix proteins in the process of shell mineralization. The eggshell matrix proteins can be divided into 3 groups according to the specificity of their expression i) some are egg white proteins, ii) others have a widespread (ubiquitous) tissue expression and finally iii) there are components whose expression is unique to the uterus and therefore to some aspect of shell formation. Antibacterial proteins of the egg white (lysozyme, ovotransferrin) are also present in the eggshell. Recently, we cloned ovocalyxin-36, a novel eggshell protein whose sequence is related to those of lipopolysaccharide binding proteins (LBP), bactericidal permeability increasing proteins (BPI) and the PLUNC family of proteins. Another novel eggshell matrix protein (Ovocalyxin-25) has been cloned and characterized as a serine protease inhibitor which could act to inhibit bacterial penetration into the egg. Chicken eggshell matrix proteins are therefore involved in the natural egg defences and preservation of the hygienic quality of eggs by at least two distinct mechanisms: regulation of shell strength by influencing mineralization, and by introducing anti-bacterial activity into the shell and its associated cuticle and membranes. Introduction In Europe, the majority (95 %) of eggs are produced in conventional cage systems. By 2012 the EU directive 1999/74 defining minimum standards for the protection of laying hens plans to abolish the conventional cage system in favour of furnished cages, aviary or other floor systems in order to improve the welfare of the hens. These modifications of the housing systems will increase the microbial risk in eggs produced for human consumption. In this context, it is important to understand and to improve the egg’s natural defences against bacteria. The egg possesses two major natural antimicrobial mechanisms. The first one is the eggshell, together with the cuticle and membranes, which constitutes a physical barrier against bacteria penetration. Eggshell is a highly organized structure whose strength depends on interactions between calcite crystals, and the organic fraction including matrix proteins and glycosaminoglycans (Nys et al. 2004). Defects in the mineralized shell are directly related to increasing risk of egg contamination. The second defence is the chemical barrier composed of proteins found in the eggshell and in the albumen that exhibit anti-microbial activity. Proteins with this property appear to be specifically and sequentially expressed throughout the length of the oviduct. The purpose of this report is to review the eggshell matrix proteins that are likely to participate in the natural defence of the egg by their anti-microbial activity. Eggshell matrix proteins The egg is sequentially formed as it traverses the oviduct of the hen. The egg white constituents are secreted by the magnum, and the shell membranes assemble on the surface of the egg white from precursors secreted in the white isthmus. Eggshell calcification is initiated in the red isthmus and then completely deposited while the egg remains in the uterus. Identification and characterization of eggshell matrix proteins is an important prerequisite to improve the eggshell quality and consequently to reinforce the natural defences of the egg. The matrix components have been intensively investigated during the past decade, and identified in decalcified shell and in the uterine fluid where XI th European Symposium on the Quality of Eggs and Egg Products Doorwerth, The Netherlands, 23-26 May 2005 310 Eggshell matrix proteins related to antibacterial proteins: J. Gautron et al. eggshell mineralization occurs. These previously identified matrix proteins can be divided into three groups according to their characteristics (Table 1). 1. The most abundant egg white proteins have been shown to be localized in the eggshell. Ovalbumin is localized in the mammillae of the eggshell (Hincke 1995) and is predominant at the initial stage of eggshell formation. Lysozyme (Hincke et al., 2000) and ovotransferrin (Gautron et al., 2001a) are found at highest levels in the eggshell membranes but are also seen in the basal calcified shell and in the cuticle at the eggshell surface. 2. The ubiquitous group of eggshell matrix proteins are widely expressed in other tissues. Osteopontin, a phosphorylated glycoprotein, is observed in avian bone and other nonmineralized tissues, as well as its uterine expression and eggshell localization (Pines et al., 1995). Clusterin is a secretory glycoprotein expressed in the oviduct and other chicken tissues; it is thought to function as an extracellular chaperone. It is found in chicken egg white and shell (Mann et al., 2003). Chicken clusterin was identified in uterine fluid at all 3 stages of shell calcification and in decalcified eggshell extract. Clusterin is present in the mammilary knobs, where shell calcification is initiated, but the predominant labelling was observed in the palisade layer and in the most external calcified layer. 3. The final group of matrix proteins are unique to the process of shell calcification. Ovocleidin-17 (OC-17) was the first eggshell protein purified from the shell (Hincke et al., 1995). It is a C-type lectin-like phosphoprotein of 17 kDa (Mann and Siedler, 1999) that occurs in a glycosylated (23 kDa) and non glycosylated form in the shell matrix (Mann, 1999). Ovocleidin-116 (OC-116), another eggshell-specific protein was cloned (Hincke et al., 1999) and found to be the protein core of a 120-/200- kDa eggshell dermatan sulfate proteoglycan (Carrino et al., 1997), which has also been termed ovoglycan (Fernandez et al., 2001). It is present throughout the palisade region of the calcified eggshell. Ovocalyxin-32 (OCX-32), a 32 kDa uterine-specific protein is predominately found in the upper part of the calcified shell (cuticle, vertical crystal layer and outer palisade layer) (Gautron et al., 2001b; Hincke et al., 2003). OCX-32 was detected in uterine fluid collected at the active and terminal phase of shell calcification and in extracts from decalcified eggshell.. Database searching with the predicted protein sequence demonstrated homology to 2 distinct mammalian proteins, each with ~30% identity to OCX-32 after alignment. One is latexin, a carboxypeptidase inhibitor restricted to a subset of neurons and a few nonneural tissues (Hatanaya et al., 1994; Normant et al., 1995, Uratani et al., 2000). The other one is the putative translation product of a tazarotene-induced gene 1 (TIG1), a retinoic acid receptor -responsive gene. (Duvic et al., 1997; Nagpal et al., 1996). The functional relationship of these mammalian proteins to OCX-32 is not clear. Table 1 Identified eggshell matrix proteins. Egg white proteins Ovalbumin Lysozyme Ovotransferrin ----------------------------------------------------------------Ubiquitous proteins Osteopontin Clusterin ----------------------------------------------------------------Specific proteins Ovocleidin-116 Ovocalyxin-36 Ovocalyxin-32 Ovocalyxin-25 Ovocleidin-17 Recently, Ovocalyxin-36, corresponding to a 36 kDa component of the uterine fluid and eggshell, has been cloned. It is secreted by uterine tubular gland cells and mainly localized in the eggshell membranes (Gautron et al., 2005). OCX-36 mRNA expression was 17-fold higher in uterine tissue collected during eggshell formation, compare to the period when no shell was present. Finally, two XI th European Symposium on the Quality of Eggs and Egg Products Doorwerth, The Netherlands, 23-26 May 2005 311 Eggshell matrix proteins related to antibacterial proteins: J. Gautron et al. additional proteins (Ovocalyxin-25 and Ovocalyxin-21) have also been cloned and identified as novel eggshell matrix proteins (Gautron, Murayama, Hincke, Nys, in preparation). Antibacterial activities of eggshell matrix proteins Eggshell is not a suiTable medium for microbial growth, in part due to its physical characteristics (solid structure with low moisture content). In addition, some egg white proteins, well known for their antimicrobial properties, have been identified in egg shell which may explain some of the antimicrobial activity observed in eggshell extracts (Mine et al., 2003). Lysozyme is a key factor of the egg natural defence system against bacterial aggression in the egg white and is also found in the shell. It is well known for its anti-bacterial properties, due to its ability to hydrolyse 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycans of Gram-positive bacteria. Ovotransferrin is another major protein of egg white that is also an eggshell component; it is involved in iron transport and plays an important role in preventing growth of Gram-negative spoilage bacteria in egg white (Garibaldi, 1970). Ovotransferrin can also interact with the membrane of other Gram-negative bacteria (Valenti et al., 1983) as well as some gram-positive bacteria (Ibrahim et al., 1998). Data base searching for ovocalyxin-36 protein sequence using Blastp showed significant identities with lipopolysaccharide binding proteins (LBP) (Schumann et al., 1990), bactericidal permeability increasing proteins (BPI) (Gray et al., 1989) and the PLUNC family of proteins (Bingle and Craven, 2002). LBP/BPI and PLUNC proteins are often described as "first-line host defence proteins" and it has been reported that they could be involved in the innate immune response. Members of the LBP/BPI family are proteins which bind to the lipid A portion of lipolysaccharide (LPS) cell wall in gram negative bacteria, leading to death of the bacterium. This interaction could be promoted in OCX-36, since PROSITE analysis (http://www.ewpasy.org/tools/scanprosite) of its amino acid sequence identifies a PROKAR_LIPOPROTEIN motif. While the amino acid motif leading to S-acylation of eukaryotic proteins remains unclear, it is possible that OCX-36 carries a lipid moiety at the N-terminus of the protein (Mattar et al., 1994). This lipid moiety could provide a mechanism for OCX-36 to interact with LPS in the cell wall of gram negative bacteria. The PLUNC family of candidate host defence proteins is expressed in the upper airways and nasopharynx of mammalian species. The complete human PLUNC gene locus is contained within approximately 300 kb on chromosome 20q11.2. The location of LBP and BPI genes are also adjacent on the same human chromosome 20q12-q13.1. PROSITE analysis of ovocalyxin-25 (OCX-25) indicated the presence of 2 domain signatures that are found in serine protease inhibitors. These are the WAP type which contains 8 cysteine residues involved in four disulfide bonds (Hennighausen and Sippel, 1982) and the Kunitz bovine pancreatic trypsin inhibitor motif (Laskowski and Kato, 1980; Salier 1990). Chelonianin is one of the models of this group protein. Our data showed that ovocalyxin-25 also exhibited these two motifs. Protease inhibitors are known to regulate activities of serine proteases. Their role in natural defence against micro-organisms is unclear but their inhibitory properties suggest that they could interfere with the activity of bacterial proteases necessary for adhesion and infectivity of the pathogen (Molla et al., 1987). Finally, the genomic localisation of Ovocalyxin-36 and Ovocalyxin-25 demonstrates that both genes are adjacent on chicken chromosome 20, as observed for the corresponding human genes. In view of this data, we propose that OCX-36 and OCX-25 could be novel chicken eggshell matrix proteins with anti-bacterial properties. They would act either in the uterine fluid during the initial stage of eggshell formation, before the establishment of the shell as a protective barrier, or at oviposition, when the eggshell still contains some moisture. Conclusion The chicken eggshell is a sophisticated structure which contains a number of proteins that have been identified and characterized during the past decade. Our experimental data shows that these proteins regulate shell mineralization and may influence its mechanical properties. These matrix proteins could also act as anti-bacterial agents to protect the egg contents against microbial challenge. 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