Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene; new insights into the function of the protein

Abstract

Whey acidic protein (WAP) belongs to a family of four disulfide core (4-DSC) proteins rich in cysteine residues and is the principal whey protein found in milk of a number of mammalian species. Eutherian WAPs have two 4-DSC domains, whereas marsupial WAPs are characterized by the presence of an additional domain at the amino terminus. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. We have characterized the genomic structure of tammar WAP (tWAP) gene, identified its chromosomal localization and investigated the potential function of tWAP. We have demonstrated that tWAP and domain III (DIII) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of DIII from tWAP abolishes this proliferative effect. However, tWAP does not induce proliferation of human embryonic kidney (HEK293) cells. DNA synthesis and expression of cyclin D1 and cyclin-dependent kinase-4 genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DIII. These data suggest that DIII is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy.

Evolution of Lactation: Ancient Origin and Extreme Adaptations of the Lactation System

“…A detailed analysis of the structure of WAP has shown extensive rearrangements of the genes in mammalian lineages, leading to a reorganization of the number of exons from monotremes to marsupials, whereas a functional gene has been lost in human, cow, and goats (84). Preliminary experiments suggest that specific WAP domains carry unique functional activities in different lineages (93).…”

“…A number of whey proteins such as alpha-lactalbumin, lactotransferin, and WAP and WDC2 have been identified. WAP has shown extensive rearrangements in mammalian lineages and WAP domains might carry specific functional activities in different lineages (93). However, the function of WAP is not fully understood.…”

“…Recently, genomics and transcriptomics approaches have enabled the molecular analysis of monotreme (48), marsupial (47), and eutherian (49) lactation. Comparative approaches have started to allow a detailed analysis of the functional evolution of specific molecular components of lactation (59,93); these results are reviewed here.…”

The evolution of milk secretion and its ancient origins

“…The structural similarity of WAP to other WFDC-containing proteins has led to speculation that WAP may also have antibacterial or proteinase inhibition functions, but attempts to demonstrate this have failed (Hajjoubi et al, 2006;Sharp et al, 2007). There is evidence that WFDC domains influence cell proliferation and growth in vitro and in transgenic mice (reviewed by Topcic et al, 2009), but when the WAP gene is deleted in knockout mice, the mice continue to develop normal mammary glands indicating that WAP is not essential for mammary cell differentiation or proliferation (Triplett et al, 2005). The primary effect of WAP deletion in mice appears to be growth retardation of the young during the second half of lactation.…”

“…Of the two WFDC domains in WAP in eutherian milks, designated as DI and DII, only DII retains the characteristic N-terminal motif found in most WFDC domains (Lys-X-Gly-X-Cys-Pro, where X represents various amino acids); amino acid substitutions in this and other areas of DI may have altered the charge distribution, glycosylation sites, and conformation in such a way that original functions are no longer possible (Ranganathan et al, 1999). Monotreme and marsupial WAPs contain two to three WFDC domains, but of differing sequence and arrangement than eutherian WAPs (Sharp et al, 2007), and it is thought that they may retain functions lost in eutherians, but more evidence is required (Topcic et al, 2009). At least some eutherians have lost WAP in entirety.…”