WFDC2 is differentially expressed in the mammary gland of the tammar wallaby and provides immune protection to the mammary gland and the developing pouch young
Highlights
► WFDC2 was expressed during pregnancy, early lactation, late lactation and involution. ► WFDC2 had antibacterial activity to Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa. ► The antibacterial activity resides within domain II of WFDC2. ► WFDC2 may reduce mastitis in the mammary gland and protect the gut of the young.
Introduction
WFDC2 is also known as human epididymis 4 (HE4) and is part of a large family of WAP four disulfide core (WFDC) proteins (Bingle et al., 2002). Proteins that belong to this family all contain four-disulfide core (4-DSC) domains that consist of eight cysteine residues in a conserved arrangement (Topcic et al., 2009, Hennighausen and Sippel, 1982, Campbell et al., 1984). Tammar wallaby (Macropus eugenii) WFDC2 has two 4-DSC domains that have previously been annotated domain III on the amino terminal end and domain II at the carboxyl terminal end based on similarities to the annotated whey acidic protein (WAP) domains (Sharp et al., 2007). WFDC2 was first identified in epididymis but was later found to have high expression in the oral cavity (Kirchhoff et al., 1991, Bingle et al., 2002, Idoji et al., 2008) and more recently the WFDC2 gene has been identified in the mammary gland of the tammar wallaby during lactation (Sharp et al., 2007).
In the tammar wallaby milk composition changes throughout lactation in order to meet the developmental requirements of the growing young (Trott et al., 2003, Green et al., 1988). The lactation cycle in the tammar wallaby has been divided into four phases (1, 2A, 2B and 3) which have been defined by changes in milk composition and growth and sucking patterns of the young (Tyndale-Biscoe and Janssens, 1988, Nicholas et al., 1997, Green et al., 1988). Phase 1 consists of 26 days of pregnancy and parturition, this precedes the start of lactation (Nicholas et al., 1997). Phase 2A consists of the first 100–120 days of lactation when the young is permanently attached to the teat and in the pouch (Lefevre et al., 2007, Trott et al., 2003, Simpson et al., 2000). During this phase the volume of milk produced is low and dilute containing high levels of complex carbohydrates and low levels of protein and fat (Nicholas et al., 1997). In phase 2B the young is no longer permanently attached to the teat but remains in the pouch and sucking is less frequent. This phase usually lasts for the next 100 days (Lefevre et al., 2007, Nicholas et al., 1997, Trott et al., 2003) and the levels of protein and fat in milk remain low and carbohydrate levels remain high. Growth of the young is slow in these two phases but there is considerable development of a competent immune system (Simpson et al., 2000, Tyndale-Biscoe and Janssens, 1988). The onset of phase 3 is correlated with an increase in growth of the mammary gland and larger volumes of milk being produced that corresponds to an increased growth rate of the young (Nicholas et al., 1997). This phase begins at approximately 200 days after parturition and represents the time when the young begins to leave the pouch, and ends approximately at 350 days (Nicholas et al., 1997). In this phase there is a dramatic change in milk composition, including reduced carbohydrate concentration and increased fat and protein concentrations (Trott et al., 2003, Nicholas et al., 1997, Tyndale-Biscoe and Janssens, 1988). The composition of the major milk proteins change progressively throughout the lactation cycle (Nicholas et al., 1997). However, not all milk proteins have been characterized for expression pattern in the tammar lactation cycle and their function in either the mammary gland or neonate remains unknown.
Our previous studies have shown that the WFDC2 gene is expressed in the mammary gland of the tammar wallaby and appeared to be differentially regulated during lactation. The expression profile of the WFDC2 gene in the tammar wallaby mammary gland was shown to have high expression during phase 1 (pregnancy), a lower level of expression during phase 2A (early lactation) and no expression in Phase 2B to 3 (mid to late lactation) (Sharp et al., 2007). However, a more extensive expression profile is needed to show precise changes in WFDC2 gene expression levels during the lactation cycle. In addition, WFDC2 was not investigated in the mammary gland during involution, a time when the mammary gland undergoes significant remodeling to return to a mammary morphology that resembles a mature virgin-like structure (Li et al., 1997).
There is limited information on the biological activity of the WFDC proteins, with the exception of SLPI (Zhu et al., 2002, Hiemstra et al., 1996), Elafin (Tomee et al., 1997) and Eppin (Yenugu et al., 2004) which have been shown to have antibacterial activity as well as other functions such as anti-inflammatory, anti-viral and anti-proteinase (Moreau et al., 2008, Williams et al., 2006). Whey acidic protein (WAP) is another WFDC family member that is found in milk in some species including the tammar wallaby (Simpson et al., 2000, Topcic et al., 2009), and recently rat WAP was shown to have antibacterial activity against Staphylococcus aureus but not Escherichia coli, suggesting selectivity of antibacterial activity (Iwamori et al., 2010).
There is very limited information about the WFDC2 gene in the tammar wallaby and WFDC2 has not been identified in the milk of other species except for the platypus and echidna (Sharp et al., 2007). The role of WFDC2 is unknown, but it has been suggested that WFDC2 in the human may play a role in innate immune defense in the lung and oral cavity (Bingle et al., 2006).
Here, we have presented an extensive expression profile of the WFDC2 gene in the mammary gland throughout the lactation cycle and in involution of the tammar wallaby. We also present functional studies which suggest that WFDC2 has a role in innate immunity by providing antibacterial activity against S. aureus, Salmonella enterica and Pseudomonas aeruginosa and that this activity is found exclusively in domain II of the two 4-DSC domains.
Section snippets
Phylogenetic analysis of WFDC2 and its 4-DSC domains
Phylogenetic relationships of WFDC2 proteins between species were performed using either the whole protein sequence or a single 4-DSC domain sequence. Sequences were aligned using the ClustalW program (Thompson et al., 1994) to determine any similarities between the WFDC2 proteins and between the 4-DSC domains of WFDC2 protein. One hundred bootstrap replicates were performed on the aligned protein sequences using seqboot followed by Protdist, a program to measure distances of protein sequences (
Phylogenetic analysis of the WFDC2 protein
To determine the evolutionary relationships between WFDC2 orthologues a phylogenetic analysis of WFDC2 proteins from different taxa was performed (Fig. 1A). This analysis showed WFDC2 proteins from marsupial (tammar wallaby and short-tailed opossum) clustered together within the same clade while WFDC2 protein from monotremes (platypus and echidna) formed a sister clade.
Classification of the type of 4-DSC domain (DIII or DII) represented in the tammar wallaby WFDC2 protein was analyzed using
Discussion
Milk has a major role in providing appropriate nutrition for the developing young but is now apparent that milk includes a range of bioactives that also have a role in regulating development of the young and promoting function and protection of the mammary gland (Piper et al., 2007, Baldi et al., 2005). The outcomes of the current study are consistent with WFDC2 having multiple roles in both the young and the mammary gland and the unique reproductive strategy of the marsupial better allows an
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