Molecular evolution of monotreme and marsupial whey acidic protein genes

Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.     

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

SUMMARY 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.     

WAP domain proteins as modulators of mucosal immunity

WAP (whey acidic protein) is an important whey protein present in milk of mammals. This protein has characteristic domains, rich in cysteine residues, called 4-DSC (four-disulfide core domain). Other proteins, mainly present at mucosal surfaces, have been shown to also possess these characteristic WAP-4-DSC domains. The present review will focus on two WAP-4-DSC containing proteins, namely SLPI (secretory leucocyte protease inhibitor) and trappin-2/elafin. Although first described as antiproteases able to inhibit in particular host neutrophil proteases [NE (neutrophil elastase), cathepsin-G and proteinase-3] and as such, able to limit maladaptive tissue damage during inflammation, it has become apparent that these molecules have a variety of other functions (direct antimicrobial activity, bacterial opsonization, induction of adaptive immune responses, promotion of tissue repair, etc.). After providing information about the 'classical' antiproteasic role of these molecules, we will discuss the evidence pertaining to their pleiotropic functions in inflammation and immunity.     

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.     

Human Epididymis Protein-4 (HE-4): A Novel Cross-Class Protease Inhibitor

Epididymal proteins represent the factors necessary for maturation of sperm and play a crucial role in sperm maturation. HE-4, an epididymal protein, is a member of whey acidic protein four-disulfide core (WFDC) family with no known function. A WFDC protein has a conserved WFDC domain of 50 amino acids with eight conserved cystine residue. HE-4 is a 124 amino acid long polypeptide with two WFDC domains. Here, we show that HE-4 is secreted in the human seminal fluid as a disulfide-bonded homo-trimer and is a cross-class protease inhibitor inhibits some of the serine, aspartyl and cysteine proteases tested using hemoglobin as a substrate. Using SPR we have also observed that HE-4 shows a significant binding with all these proteases. Disulfide linkages are essential for this activity. Moreover, HE-4 is N-glycosylated and highly stable on a wide range of pH and temperature. Taken together this suggests that HE-4 is a cross-class protease inhibitor which might confer protection against microbial virulence factors of proteolytic nature.     

Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells

Whey acidic protein (WAP) is the principal whey protein found in rodent milk, which contains a cysteine-rich motif identified in some protease inhibitors and proteins involved in tissue modeling. The expression of the Wap gene, which is principally restricted to the mammary gland, increases more than 1,000-fold around mid-pregnancy. To determine whether the expression of this major milk protein gene is a prerequisite for functional differentiation of mammary epithelial cells, we generated conventional knockout mice lacking two alleles of the Wap gene. Wap-deficient females gave birth to normal litter sizes and, initially, produced enough milk to sustain the offspring. The histological analysis of postpartum mammary glands from knockout dams does not reveal striking phenotypic abnormalities. This suggests that the expression of the Wap gene is not required for alveolar specification and functional differentiation. In addition, we found that Wap is dispensable as a protease inhibitor to maintain the stability of secretory proteins in the milk. Nevertheless, a significant number of litters thrived poorly on Wap-deficient dams, in particular during the second half of lactation. This observation suggests that Wap may be essential for the adequate nourishment of the growing young, which triple in size within the first 10 days of lactation. Important implications of these findings for the use of Wap as a marker for advanced differentiation of mammary epithelial cells and the biology of pluripotent progenitors are discussed in the final section.     

Whey acidic protein (WAP) regulates the proliferation of mammary epithelial cells by preventing serine protease from degrading laminin

Whey acidic protein (WAP) is a major whey protein in milk that has structural similarity to the family of serine protease inhibitors with WAP motif domains characterized by a four-disulfide core. We previously reported that enforced expression of the mouse WAP transgene in mammary epithelial cells inhibits their proliferation in vitro and in vivo by means of suppressing cyclin D1 expression (Nukumi et al., 2004, Dev Biol 274: 31-44). This study was conducted in order to clarify the molecular mechanism of the inhibitory function of WAP in HC11 cells, a mammary epithelial cell line. The assembly of laminin, a component in the extracellular matrix, was much more prominent around WAP-clonal HC11 cells that stably expressed the WAP transgene than around mock-clonal HC11 cells, and the proliferation of WAP-clonal HC11 cells was particularly inhibited in the presence of laminin. A laminin degradation assay demonstrated that WAP inhibited the activity of the pancreatic elastase-mediated cleavage of laminin B1 and the phosphorylation of ERK1/2. ERK1/2 phosphorylation was blocked by an inhibitor of the epidermal growth factor (EGF) receptor AG1478. Treatment with pancreatic elastase was found to enhance the proliferation of mock-clonal HC11 cells, but had no effect on that of WAP-clonal HC11 cells. The proliferation of WAP-clonal HC11 cells was recovered by the addition of exogenous EGF. We concluded that WAP plays some role in regulating the proliferation of mammary epithelial cells by preventing elastase-type serine protease from carrying out laminin degradation and thereby suppressing the MAP kinase signal pathway.     

A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis

The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78 kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillus subtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens.     

Phylogeny of whey acidic protein (WAP) four-disulfide core proteins and their role in lower vertebrates and invertebrates

Proteins containing WAP (whey acidic protein) domains with a characteristic WFDC (WAP four-disulfide core) occur not only in mammals (including marsupials and monotremes) but also in birds, reptiles, amphibians and fish. In addition, they are present in numerous invertebrates, from cnidarians to urochordates. Many of those from non-mammalian groups are poorly understood with respect to function or phylogeny. Those well characterized so far are waprins from snakes, perlwapins from bivalves and crustins from decapod crustaceans. Waprins are venom proteins with a single WAP domain at the C-terminus. They display antimicrobial, rather than proteinase inhibitory, activities. Perlwapins, in contrast, possess three WAP domains at the C-terminus and are expressed in the shell nacre of abalones. They participate in shell formation by inhibiting the growth of calcium crystals in the shell. The crustin group is the largest of all WFDC-containing proteins in invertebrates with the vast majority being highly expressed in the haemocytes. Most have a single WAP domain at the C-terminus. The presence and type of the domains between the signal sequence and the C-terminus WAP domain separate the different crustin types. Most of the Type?I and II crustins are antimicrobial towards Gram-positive bacteria, whereas the Type?III crustins tend to display protease inhibition. Expression studies show that at least some crustins have other important biological effects, as levels change with physiological stress, wound repair, tissue regeneration or ecdysis. Thus WAP domains are widely distributed and highly conserved, serving in diverse physiological processes (proteinase inhibition, bacterial killing or inhibition of calcium transport).     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Characterization of two whey protein genes in the Australian dasyurid marsupial, the stripe-faced dunnart (Sminthopsis macroura)

We report the first isolation and sequencing of genomic BAC clones containing the marsupial milk protein genes Whey Acidic Protein (WAP) and Early Lactation Protein (ELP). The stripe-faced dunnart WAPgene sequence contained five exons, the middle three of which code for the WAPmotifs and four disulphide core domains which characterize WAP. The dunnart ELPgene sequence contained three exons encoding a protein with a Kunitz motif common to serine protease inhibitors. Fluorescence in situ hybridization located the WAPgene to chromosome 1p in the stripe-faced dunnart, and the ELPgene to 2q. Northern blot analysis of lactating mammary tissue of the closely related fat-tailed dunnart has shown asynchronous expression of these milk protein genes. ELPwas expressed at only the earlier phase of lactation and WAPonly at the later phase of lactation, in contrast to beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) genes, which were expressed in both phases of lactation. This asynchronous expression during the lactation cycle in the fat-tailed dunnart is similar to other marsupials and it probably represents a pattern that is ancestral to Australian marsupials.     

Functional studies of eppin

Our laboratory has characterized EPPIN [epididymal protease inhibitor; SPINLW1] as a novel gene on human chromosome 20q12-13.2, which encodes a cysteine-rich protein of 133 amino acids with a calculated molecular mass of 15.283?kDa, containing both Kunitz-type and WAP (whey acidic protein)-type four-disulfide core consensus sequences. Eppin is secreted by Sertoli cells in the testis and epididymal epithelial cells; it is predominantly a dimer, although multimers often exist, and in its native form eppin is found on the human sperm surface complexed with LTF (lactotransferrin) and clusterin. During ejaculation SEMG (semenogelin) from the seminal vesicles binds to the eppin protein complex, initiating a series of events that define eppin's function. Eppin's functions include (i) modulating PSA (prostate-specific antigen) enzyme activity, (ii) providing antimicrobial protection and (iii) binding SEMG thereby inhibiting sperm motility. As PSA hydrolyses SEMG in the ejaculate coagulum, spermatozoa gain progressive motility. We have demonstrated that eppin is essential for fertility because immunization of male monkeys with recombinant eppin results in complete, but reversible, contraception. To exploit our understanding of eppin's function, we are developing compounds that inhibit eppin-SEMG interaction and mimic anti-eppin, inhibiting sperm motility. These compounds should have potential as a male contraceptive.     

Cloning, transcription and chromosomal localization of the porcine whey acidic protein gene and its expression in HC11 cell line

The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine (Simpson et al., 1998. J. Mol. Endocrinol. 20, 27-35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.     

Transgenic rabbit producing human growth hormone in milk

The gene construct WAP(6xHisThr):hGH containing the entire human growth hormone gene (hGH) under the rat whey acidic protein (WAP) promoter regulating the expression in mammary glands of mammals was prepared. The 5' end of the gene was modified by the addition of a sequence encoding six histidine residues and a sequence recognized by thrombin. The gene construct was introduced by microinjection into the male pronucleus of a fertilized oocyte. The founder male rabbit was obtained with the transgene mapping to chromosome 7. The presence of the growth hormone was confirmed in samples of milk collected during the lactation of F1 generation females. The growth hormone can be easily purified by affinity chromatography and cleavage by thrombin to an active form.     

Secretion of unprocessed human surfactant protein B in milk of transgenic mice

Because of the apparent clinical importance of human pulmonary surfactant B (SP-B), the expression of SP-B was directed to the mammary gland of transgenic mice using previously characterized rat whey acidic protein (WAP) regulatory sequences. rWAP/SP-B mRNA was expressed specifically in the mammary gland, and ranged from 1 to 5% of the endogenous WAP mRNA levels. SP-B was detected immunologically in both tissue and milk. The transgene product had an apparent molecular weight of 40--45 kDa, corresponding to the predicted size of the SP-B proprotein. Incubation of an SP-B-enriched fraction of milk with cathepsin D in vitro produced 20--25 kDa species, consistent with cleavage of the amino terminal domain by cathepsin D. This was confirmed using antibodies specific to the carboxy-terminal domain of SP-B. However, the appearance of only the SP-B proprotein in milk suggests that cathepsin D is not involved in the in vivo processing of SP-B. The SP-B proprotein can be expressed in milk of transgenic mice without any observed effects on mammary gland morphology or lactation     

Use of the urokinase-type plasminogen activator gene as a general tool to monitor expression in transgenic animals: study of the tissue-specificity of the murine whey acidic protein (WAP) expression signals.

Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme able to convert the zymogen plasminogen into the strong protease plasmin. The availability of very sensitive tests to measure the enzymatic activity of a plasminogen activator renders the corresponding gene an ideal candidate for the detection of promoter activity. In this paper we describe the utilization of the human uPA gene as detector of tissue-specificity of the murine whey acidic protein (WAP) expression signals in transgenic mice. The WAP promoter has been previously investigated for the production of foreign proteins in the milk of transgenic animals. In our genetic constructions, the human uPA cDNA was linked to the promoter region as well as to 3'-end distal sequences of the WAP gene. Five transgenic lines were obtained in which, however, expression levels of human uPA in the milk were still quite low. Surprisingly, four of these five positive transgenic mice show a consistent activity of the WAP promoter in brain extracts compared to other tissues.