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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
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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
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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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Acknowledgements
We thank the European commission (egg defence, QLRT-2001-01606), NSERC and the Poultry
Industry Council for financial support of some of this work. We are grateful to M. Peloille (cDNA
sequencing), and to A.P. Teixeira (microsequencing) at INRA center, Tours, France and to the region
center for financial support of the sequencing work-shop and apparatus.
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