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Originally published In Press as doi:10.1074/jbc.M002161200 on May 3, 2000

J. Biol. Chem., Vol. 275, Issue 30, 23074-23081, July 28, 2000
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The Gene for a Novel Member of the Whey Acidic Protein Family Encodes Three Four-disulfide Core Domains and Is Asynchronously Expressed during Lactation*

Kaylene J. SimpsonDagger §, Shoba Ranganathan||, Juliet A. Fisher**DaggerDagger, Peter A. Janssens**, Denis C. Shaw§§, and Kevin R. NicholasDagger

From the Dagger  Victorian Institute of Animal Science, 475 Mickleham Rd., Attwood, Victoria 3049, the § School of Agricultural Sciences, La Trobe University, Bundoora, Victoria 3083, the || Australian Genomic Information Centre, C80 ATP, University of Sydney, Sydney, New South Wales 2006, the ** Division of Biochemistry and Molecular Biology, Australian National University, Canberra, Australian Capital Territory 2602, and the §§ Protein Biochemistry Group, John Curtin School of Medical Research, Australian National University, Australian Capital Territory 2601, Australia

Received for publication, March 15, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of the tWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Whey acidic protein (WAP)1 has been identified in the milk of a limited number of species and is the major whey protein in the mouse (1), rat (2), rabbit (3), and camel (4). It is also a significant component of porcine milk (5). The WAP proteins from all species show limited sequence identity at the amino acid level but are recognized by a two-domain structure, known as the four-disulfide core (4-DSC) (6) domain, which comprises eight cysteine residues in a conserved arrangement (1). The 4-DSC domain arrangement is not exclusive to the WAP family of proteins; numerous other proteins have been identified that contain one or two such domains. Although a large biological diversity exists between these proteins, many have been identified as protease inhibitors and are grouped into families based on their functionality and tissue-specific origins. These families include the antileukoproteinase (ALKI) family (7), epididymal (8) and ovulatory (9) specific proteins, and elastase inhibitor proteins (10). Given such a variety of proteins, it is thought that the 4-DSC may be a preferential conformation for the stable folding and action of a particular class of protease inhibitors (6, 11). Despite no biological action or function being demonstrated for the WAP proteins and based on the limited sequence identity with known protease inhibitors, it has been postulated that WAP may be a protease inhibitor (6, 12).

We have reported previously (13) that WAP is present in the milk of a marsupial, the tammar wallaby (Macropus eugenii), and preliminary data showed the secretion of this protein was developmentally regulated during the lactation cycle. The tammar wallaby is widely used as a model to study marsupial development, and as a result its reproduction cycle has been well defined (14-16). This species has adopted a comparatively different reproductive strategy to other eutherians, i.e. a short gestation and birth of an immature young followed by a relatively long lactation (14, 15). Milk is the only nutritional source available to the rapidly developing pouch young until it has reached physiological maturity and is able to move outside the pouch and eat herbage (14). The composition of the milk is complex, and it changes during the lactation cycle to meet the nutritional demands of the pouch young. The lactation cycle in the tammar has been divided into four distinct phases (17), which are characterized by both changes in milk composition and changes in the sucking pattern of the pouch young. Phase 1 comprises the 26.5-day gestation period during which time all four mammary glands prepare for parturition and the onset of lactogenesis (18). At parturition, a single immature young climbs into the pouch and attaches to one of the four available teats, and remains permanently attached to this teat for the next 100-120 days (phase 2A (17)), receiving a limited volume of milk that is low in fat and protein but high in complex carbohydrates (19, 15). At the onset of phase 2B at around days 100-120, the pouch young relinquishes the teat but remains in the pouch for a further 80-100 days. During phase 2A and 2B the rate of growth is slow, with a large component of the milk nutrients utilized for development of physiological functions (14). The commencement of phase 3 (day 200) is characterized by an increase in milk production and size of the mammary gland, together with the secretion of a more concentrated milk, which is high in lipid and protein and low in carbohydrate (19, 15). The growth rate of the pouch young accelerates, and at approximately day 250 it permanently leaves the pouch, adopting a diet of milk and herbage, until weaning at 300-350 days postpartum (19, 15).

We report here for the first time the isolation and purification of WAP from a marsupial, the tammar wallaby, and show that gene expression is developmentally regulated throughout the lactation cycle and appears potentially controlled by a novel signaling mechanism intrinsic to the mammary gland. The structure of tWAP does not conform to the two-domain structure described for WAPs from all other species. A consensus three-dimensional structural model for the two-domain eutherian WAP proteins was developed recently, with a detailed atomic model created for pig WAP (pWAP (6)). This paper reports an analogous three-domain structural model for tWAP, and together with the unique profile for developmentally regulated expression of the gene for tWAP, provides new opportunities to examine the role of this protein during lactation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All tissue culture reagents were purchased from Life Technologies, Inc. Ovine prolactin (NIH-oPRL-16) was provided by National Hormone and Pitvitary Program, University of Maryland, School of Medicine (Baltimore, MD). Bovine insulin, hydrocortisone, tri-iodothyronine, and estradiol were purchased from Sigma.

Housing of Animals-- Tammars were maintained on site at the Victorian Institute of Animal Science in a natural habitat with access to water and feed supplements ad libitum.

Purification of tWAP by HPLC and Amino Acid Sequence Analysis-- Acidified whey from various stages of lactation was fractionated by high performance liquid chromatography (HPLC) (5) using a POROS R2/H reverse-phase column and a 15-60% gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 4 ml/min. The absorbance of proteins eluting from the column was measured at both 215 and 280 nm, and fractions were dried under vacuum and stored at 4 °C. Proteins were resuspended in distilled water, analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and identified by N-terminal amino acid sequencing using a Procise 494 protein sequencer.

SDS-PAGE Analysis of Tammar Milk Proteins-- Whole milk was collected from tammar wallabies (15) at various stages throughout lactation. Skim milk was separated from fat and cell debris by centrifugation at 2000 × g for 10 min at 4 °C, and the casein fractions were pelleted by centrifugation at 100,000 × g for 30 min at 12 °C. Diluted whey samples were separated in 20% polyacrylamide gels under reducing conditions (20) and stained with Coomassie Blue R-250.

Isolation of tWAP cDNA-- An amplified cDNA library made from RNA from tammar wallaby mammary gland at day 201 of lactation cloned in the EcoRI site of phage gt-11 was grown in log phase Y1090 culture (including tetracycline selection (12.5 µg/ml), maltose (0.2% v/v), and MgSO4 (10 mM)). Plaques (40,000) were screened with an end-labeled degenerate antisense oligonucleotide designed to known tWAP amino acid sequence (residues 24-31; tWAP 1, 5'-GCRTCRTTRCANAARYTGYTTRCA-3'). Four plaques were analyzed through secondary and tertiary screens, the lambda DNA was digested with EcoRI, and the insert fragment was cloned into the pGEM-7 vector (Promega). The clone was sequenced from both directions using T7 and SP6 primers and dye terminator chemistry (Applied Biosystems).

tWAP Gene Expression During Lactation-- Total RNA was extracted (21) from the mammary glands of each of three lactating tammars at intervals from day 95 to day 276 of lactation. Each sample (10 µg) was fractionated through a 1.4% agarose formaldehyde gel and transferred to a Zeta Probe GT membrane (Bio-Rad). The tWAP and tammar beta -lactoglobulin (beta -LG) cDNAs were hybridized with the membranes (5), washed at 60 °C to high stringency (0.1% SSC/0.1% SDS), and exposed to Kodak Xomat film at -70 °C. The tWAP mRNA levels were quantitated by slot blot analysis (22). The membranes were hybridized with the tWAP cDNA and washed to high stringency, and each individual slot was excised and placed into 5 ml of organic scintillant. The radioactivity was measured in a liquid scintillation spectrophotometer (Wallac 1410, Amersham Pharmacia Biotech).

Mammary Gland Explant Culture System-- Mammary gland tissue from tammars during late pregnancy (phase 1, days 23-25), phase 2B (day 180), and phase 3 (day 260) of lactation were cut into explants of approximately 1-2 mg and cultured on floating siliconized lens paper in media 199 (M199) supplemented with the indicated combinations of bovine insulin (I, 1 µg/ml), cortisol (F, 0.05 µg/ml), ovine prolactin (P, 0.2 µg/ml), tri-iodothyronine (T3, 6.5 pg/ml), and estradiol (E2, 1 pg/ml). Media was changed daily. Mammary tissue was collected for analysis prior to culture (T0), and explants from two dishes (approximately 40 explants) were collected and pooled for each treatment after 4, 8, and 12 days of culture. Total RNA was extracted from the tissue using Trizol reagent (Life Technologies, Inc.) and fractionated through a 1.4% agarose formaldehyde gel, transferred to Zeta Probe GT membrane, and hybridized with the tWAP and beta -LG cDNAs using the conditions described previously.

Sequence Alignment and Three-dimensional Model Building-- The WAP sequences were aligned with other 4-DSC protein sequences initially using Clustal-W. However, due to the limited sequence identity, the alignment was edited visually to align the eight cysteine residues of each domain. The mature tWAP sequence was aligned with the sequence of recombinant human secretory leukocyte proteinase inhibitor (hSLPI), which has been shown to contain two 4-DSC domains (24). The program Modeller (25) was used to generate the tWAP structure, specifying the formation of four disulfide bridges in each domain, and subjecting the model to iterative molecular dynamics refinement using built-in simulated annealing protocols (6).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of tWAP by HPLC-- HPLC fractionation of tammar whey collected at 14- to 21-day intervals from days 88 to 270 of lactation resulted in an average of 10 major protein peaks, depending on the stage of lactation (Fig. 1). Examination of eluates at an absorbance of 215 nm showed that several peaks either appeared or disappeared from the milk at specific times during the course of lactation. Peaks 2 and 3 were first detected at approximately day 116, reaching a maximum around day 193 (Fig. 1) and then reducing to undetectable levels by day 256 of lactation (data not shown). The protein component of these peaks, eluting as a doublet at 29.5% and 30.6% acetonitrile (Fig. 1), was subjected to tryptic digestion and N-terminal amino acid sequencing and revealed 46 residues, which shared significant sequence identity with camel WAP (cWAP). The protein obtained from peak 2 at day 193 of lactation was analyzed by 20% SDS-PAGE and showed tWAP migrated as a major protein at 29.9 kDa (Fig. 1, inset).


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  		Fig. 1.   Identification of tWAP by reverse-phase HPLC. The HPLC profile of whey from day 193 of lactation measured at an absorbance of 215 nm shows 10 major protein peaks. tWAP (indicated by the arrow) was detected in peaks 2 and 3, eluting as a doublet at approximately 29% acetonitrile. The protein component of peak 2 was analyzed by 20% SDS-PAGE and is shown in the inset, with tWAP migrating as a major band at 29.9 kDa. Molecular mass markers are indicated in kDa.

The Profile of tWAP Secretion during Lactation-- SDS-PAGE analysis of total whey from day 140 to 276 of lactation showed the asynchronous changes in milk composition at the transition from phase 2B to phase 3 (Fig. 2). tWAP was a major whey protein at day 202 of lactation and was undetectable in phase 3 whey. Both beta -LG (18 kDa) and alpha -lactalbumin (alpha -lac; 14 kDa) were secreted throughout lactation, whereas the secretion of late lactation protein A (LLP-A; 24 kDa) and LLP-B (21 kDa) was induced around days 180 and 220 respectively, signaling the transition to phase 3 of lactation (17).


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  		Fig. 2.   SDS-PAGE analysis of tammar whole whey. The complex secretory pattern of whey proteins from various stages of lactation ranging from phase 2B (days 140-174) to phase 3 (days 202, 231, and 276). The migration of WAP and the major whey proteins beta -lactoglobulin (beta -LG), alpha -lactalbumin (alpha -lac), and late lactation proteins A and B (LLP-A and LLP-B) are indicated. Molecular mass markers are shown in kDa.

Isolation of tWAP Full-length cDNA-- A degenerate antisense oligonucleotide designed to the known tWAP peptide sequence was used to screen a cDNA library prepared from mammary gland RNA from day 201 of lactation. The clone isolated from the cDNA library was 724 base pairs (bp) and represented a full-length cDNA coding for 191 translated amino acids (GenBankTM accession number AJ005356). The translated sequence was identical to the N-terminal sequence obtained from the protein in peak 2. The cDNA contains a 23-bp 5'-untranslated region, 18-amino acid signal peptide, and 2 stop codons that are in-frame and positioned 10 amino acid residues apart (Fig. 3). The polyadenylation signal (AATAAA) is situated at nucleotide position 695, and the clone contains a further 24 bp of 3'-untranslated region but does not contain the poly(A) tail (Fig. 3). The translated sequence revealed that the tWAP gene consists of three 4-DSC domains and not the two domains reported for the eutherian WAP proteins (Figs. 3 and 4). The three domains of tWAP have been ordered consecutively as domains III, I, and II (Fig. 3), with domains I and II being conserved within other species (Fig. 4).


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  		Fig. 3.   Nucleotide and predicted amino acid sequence of the tWAP full-length cDNA clone. The tWAP full-length cDNA encodes 191 translated amino acids, comprising a hydrophobic leader peptide of 18 amino acids (dashed line), three 4-DSC domains (solid lines), two in-frame termination codons marked by asterisks (the first is indicated in boldface font), and the polyadenylation sequence is indicated in bold and underlined. The conserved cysteine residues constituting each 4-DSC domain are in bold. A single putative N-linked glycosylation site is identified at amino acids 69-71 and is indicated by the shading.


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  		Fig. 4.   Alignment of tWAP with all WAPs and selected protease inhibitors. Alignment of all WAPs with representative proteins from various families that contain one or two 4-DSC domains. The three domain structure of tWAP is shown, with domains I and II aligning with all WAPs and protease inhibitors. The conserved amino acids constituting each 4-DSC domain, represented by Cys1-(Xn)-Cys2-(Xn)-Cys3-(X5)-Cys4-(X5)-Cys5Cys6-(X3,X5)-Cys7-(X3,X4)-Cys8 (6), are indicated by black shading and white text, whereas the consensus WAP motif recognized by KXGXCP in each domain is boxed. The conserved tryptophan residue in the WAP motif of the eutherian species is also highlighted (KAGRCPW (5)). Other conserved amino acids are indicated by gray shading and bold type. The site for potential O-linked glycosylation is indicated by double lines. Gaps were introduced for maximal alignment of amino acids. Proteins were truncated at the N terminus to begin the alignment at the WAP motif for either the first or second domain. Truncation is recorded from the initiation methionine unless noted and data base accession numbers correspond to SWISS-PROT or TrEMBL (in italics). Tammar WAP (tWAP, +24), platypus WAP (platWAP, +6), echidna WAP (echWAP, +6), porcine WAP (pWAP, O46655, +27), rabbit WAP (rabWAP, P09412, +27), camel WAP (cWAP, P09837, +8), rat WAP (rWAP, P01174, +29), mouse WAP (mWAP, P01173, +29), trout ovulatory protein 1 (TOP-1, Q91450, +33), human secretory leukocyte inhibitor (hSLPI, P03973, +85), human epididymal protein (HE4, Q14508, +31), and rat WDNM1 (rWDNM1, P14730, +14).

An alignment of all WAP proteins and selected representatives from several protein families that contain one or two 4-DSC domains showed the characteristic conservation of the cysteine residues within each domain (Fig. 4). Each domain includes eight cysteine residues, recognized by the consensus pattern: Cys1-(Xn)-Cys2-(Xn)-Cys3-(X5)-Cys4-(X5)-Cys5Cys6-(X3,X5)-Cys7-(X3,X4)-Cys8 (6). Domain II is more conserved than domain I in all proteins. At the amino acid level, tWAP shares 22.9% sequence identity with mouse WAP (mWAP), 25.4% with rat WAP, 25.1% with rabbit WAP, 25.5% with cWAP and 26.6% with pWAP (Fig. 4). Limited N-terminal sequence was also available for putative WAPs from platypus (platWAP)2 and echidna (26), and these have been aligned with domain III of tWAP based on sequence identity. tWAP shares 34% identity over the first 37 amino acids with platWAP and 41% with echidna WAP over the first 51 amino acids.

In addition to the conserved cysteine residues, several proline, lysine, glutamic acid, and glycine residues are conserved at the same position in each domain (5, 6), including tWAP domain III (Fig. 4). An interesting feature of the WAP proteins is a conserved WAP motif of seven amino acids, KAGRCPW, which is present in the mouse, rat, rabbit, camel, and pig and is located at the start of domain II and includes the first cysteine residue of the domain (5). In all protease inhibitors with one or two domains, the motif is also present at the beginning of each domain, however, it is less conserved, resulting in a consensus sequence of KXGXCP (Figs. 4 and 6). This consensus motif is present at three locations in tWAP and is situated at the beginning of each domain and includes the first cysteine residue of that domain (Fig. 4). Similarly, the motif is also present in the putative domain III of the echidna and platypus WAP.

Examination of the amino acid sequence of tWAP revealed one potential site for N-linked glycosylation, characterized by the sequence Asp-X-(Ser/Thr), where X is not a proline (27). This occurs at positions 69-71, residues Asp-Thr-Thr, located near the end of domain III (Fig. 3). There are no potential N-linked glycosylation sites present in any of the other WAP proteins (Figs. 4 and 6). The consecutive series of serine residues in domain I of the WAP proteins is not conserved in domain I of other protease inhibitors (Figs. 4 and 6) and may represent a potential site for O-linked glycosylation (28) for some WAP sequences.

Proposed Three-dimensional Structure for tWAP-- The structures of domains I and II of tWAP were created by homology modeling, based on alignment of tWAP with the corresponding domains of hSLPI. Domain-wise structure comparison resulted in a root mean square deviation of 0.47 Å over 33 Calpha pairs (68%) for domain I and 1.06 Å over 45 Calpha pairs (99%) for domain II. tWAP domain III shares greater sequence identity with hSLPI domain I (36.7% compared with 27.1% with hSLPI domain II) and was thus modeled from the structure of hSLPI domain I, with a root mean square deviation of 0.83 Å over 44 Calpha pairs (92%). Of the three domains of tWAP, domain II is most similar to the template structure, with spatial conservation of all four disulfide bridges. The second disulfide bridge of domains I and III of tWAP is displaced about 30° toward the C terminus of the protein, as observed in the case of both pWAP domains. Despite the domain-wise structural similarity, there is considerable rearrangement of the packing of the three domains into a quaternary structure, compared with both the hSLPI structure and the pWAP model (6). This rearrangement is a consequence of fewer "interdomain" residues (defined as the number of amino acids between the last cysteine of one domain and the first cysteine of the next domain) in tWAP (10 each) compared with hSLPI (16) and pWAP (13, 6). The two domains of hSLPI are oriented at 150o to each other, with a translation of 16.4 Å (based on the structure comparison method of Reference 29), whereas the three tWAP domains are at about 120o with a translation of 9.5 Å between domains I and II and 12.7 Å between domains III and I. MOLSCRIPT (30) graphic representations of the tWAP model are shown in Fig. 5 (A and B). The tWAP model was assessed using PROCHECK (31) as being structurally very satisfactory, with 97.4% of its residues adopting allowed backbone conformations. The surface electrostatic potential (results not shown), computed using GRASP (32), of the top outer loop of each tWAP domain is hydrophobic, similar to the serine protease inhibitors that have a 4-DSC domain (24). The coordinates of the model structure are available from the Protein Data Bank (PDB code: 1TWP).


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  		Fig. 5.   Three-dimensional model for tWAP. The model is presented as a MOLSCRIPT picture. A, the N terminus begins as blue and the color spectrum continues to change to red at the C terminus. Spheres indicate the Calpha atoms of the first (in purple) and last (in red) residues of each 4-DSC domain. The four-disulfide bridges are shown by black bars and are labeled SS1-SS4: in blue, domain III; green, domain I; and red, domain II. The heavy atoms of each bridge, the putative N-glycosylation site (N69), and the residues occupying the "scissile bond" positions are shown in ball-and-stick representation, colored by atom type (oxygen, red; nitrogen, blue; sulfur, yellow; carbon, gray). The spirals represent alpha -helical segments, and the arrows represent beta -strands. B, view of the tWAP model along the pseudo 3-fold axis, with the putative scissile bond residues indicated and domains labeled.

The two domains of hSLPI are capable of independent function, inhibiting trypsin and chymotrypsin, respectively (24). The scissile bonds for hSLPI domain I are residues RY (residues 20 and 21) and for domain II are LM (residues 72 and 73) (24). The structurally equivalent residues in tWAP are RD (domain III: residues 40 and 41), QC (domain I: residue 95, with the Cys97 Calpha atom occupying the position of Tyr21 Calpha ) and PE (domain II: residues 141 and 142) (Fig. 5B). Preliminary studies have not shown any inhibitory activity against trypsin and chymotrypsin (data not shown).

tWAP Gene Expression Profile throughout Lactation-- Total RNA was extracted from the lactating glands of tammar wallabies at phases 2A, 2B, and 3 and examined by Northern analysis. A tWAP transcript of approximately 0.8 kilobase (kb) was present only in RNA from days 140 to 231 of lactation (Fig. 6A). No transcript was detected in RNA isolated from mammary glands during gestation or from tammar liver (data not shown). Quantitative analysis of the same RNA samples using slot blots confirmed the mRNA levels were greatest around day 200 of lactation (Fig. 6B). The tammar beta -LG transcript was detected in all samples analyzed, confirming the integrity of the RNA.


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  		Fig. 6.   Developmental expression profile and quantitation of WAP mRNA levels throughout lactation. A, Northern analysis of total RNA extracted from the mammary glands of tammars during phase 2A (days 95, 96, and 106), phase 2B (days 140, 144 (two animals), 169, 172, 174, and 198), and phase 3 (days 202 (two animals), 231, 242, 244, 275, and 276 (2 animals)) of lactation. Total RNA (10 µg) was hybridized with alpha -32P-labeled tWAP and beta -LG cDNAs. Molecular weight markers are indicated in kilobases. B, the relative levels of tWAP mRNA in the same total RNA (1 µg) analyzed for A. The mRNA levels were quantitated by slot blot analysis and expressed as cpm × 103/µg of total RNA. Values are presented as mean ± S.E. (n = 3).

Endocrine Control of tWAP Gene Expression-- To establish the endocrine requirements for tWAP gene expression, explants of mammary gland tissue from either lactating or late pregnant tammars were cultured in M199 in the presence of various hormone combinations. Examination of sections of tissue taken prior to culture and stained with hematoxylin and eosin confirmed the absence of secretory cells in the pregnant tissue and presence of expanded alveoli and lipid secretions in the lactating tissue (data not shown). The tWAP mRNA was not detected by Northern analysis in uncultured mammary gland tissue (T0) at day 24 of pregnancy but was detected after 4 days of culture in media with insulin (I), cortisol (F), prolactin (P), tri-iodothyronine (T3), and estradiol (E2) (IFPT3E2) with maximal levels observed after 8 days of culture (Fig. 7A). No tWAP mRNA was detected in explants cultured in media with either IF, P, or IFP. Expression of the beta -LG gene was induced with P alone in mammary explants from tammars at day 24 of gestation and in all analyses was not detected after 4 days of culture in IF, confirming previous studies (22).


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  		Fig. 7.   Endocrine regulation of tWAP induction in vitro. The hormonal requirements for in vitro expression of tWAP and beta -LG after explant culture of mammary glands from late pregnancy (phase 1), phase 2B, and phase 3 of lactation. Mammary gland explants were cultured in the presence of insulin and cortisol (IF); prolactin alone (P); insulin, cortisol, and prolactin (IFP); and insulin, cortisol, prolactin, tri-iodothyronine, and estradiol (IFPT3E2, abbreviated here to TE). T0 indicates tissue analyzed prior to culture. Total RNA was extracted from the cultured tissue after either 4 or 8 days of culture and 10 µg were analyzed by Northern hybridization with tWAP and beta -LG cDNA probes. Ribosomal RNA bands stained with ethidium bromide are included to indicate consistency of loading. Northern blots are representative of four animals at day 24 of pregnancy, three at day 180 of lactation, and two at day 260 of lactation. A, phase 1, day 24 of pregnancy, day 8 of culture. B, phase 2B, day 180 of lactation, day 8 of culture. C, phase 3, day 260 of lactation, day 4 of culture.

To examine whether the endocrine requirements for maintenance of tWAP gene expression were different to the requirements for induction of the gene, mammary gland tissue from days 180 and 260 of lactation were cultured under the same conditions as described above. tWAP mRNA was expressed at elevated levels in the uncultured (T0) tissue at day 180 and was maintained at similar levels after 8 days of culture in IFP and IFPT3E2, but a transcript was barely detectable in explants cultured in media with P alone (Fig. 7B). In contrast, beta -LG gene expression was maintained with P alone, and stimulated above T0 values in explants cultured in media with either IFP or IFPT3E2. At day 260 of lactation the level of tWAP mRNA was barely detectable and was not stimulated in explants after 4 days in media with IFPT3E2 (Fig. 7C). In contrast, beta -LG gene expression was elevated in mammary tissue prior to culture and maintained at reduced but high levels of expression in all treatments at days 4 (Fig. 7C) and 8 of culture (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Whey acidic protein (WAP) has previously been isolated from the milk of a range of laboratory species (1-3) and the camel (4) and pig (5). In addition, the protein has been identified by limited N-terminal sequencing of HPLC-purified protein from the milk of two monotremes, the echidna (Tachyglossus aculeatus (26)) and platypus (Ornithorhynchus anatinus3). The isolation of tWAP from the milk of a marsupial reported here confirms that the synthesis and secretion of WAP is widespread in many species that have been subjected to a range of evolutionary selection pressures.

It was originally proposed that the WAP gene arose by intragenic duplication, most likely of the more conserved domain II (33). The alignment of the N-terminal sequence of the monotreme WAPs with tWAP suggests they also contain three domains. It appears probable that they are the progenitor species and that selection during evolution has reduced the protein to two domains. Despite the diversity in the origin of proteins that comprise either single or multiple copies of a 4-DSC domain (6), generally these proteins are secreted and many have established protease inhibitor functions. For example, the two-domain arrangement of the pig, mouse, rat, rabbit, and camel WAPs is present in the antileukoproteinase (ALKI) family, with genes isolated from a variety of mucosal secretory tissues from human (7), mouse (34), and pig (35). Epididymal secretory protein homologs isolated from human (HE-4 (8)), dog (CE-4 (36)), and rabbit (BE-20 (37)) also conform to this structure, along with an ovulation-specific gene, TOP-1, isolated from the brook trout (Salvelinus fontinalis) (9). In TOP-1, the two 4-DSC domains are precisely duplicated, whereas another isoform, TOP-2, encodes three perfectly duplicated 4-DSC domains, which are interspersed with a different repeated domain (9).

The second 4-DSC WAP domain is also conserved in a range of protease inhibitors with a single 4-DSC domain such as elafin, an elastase inhibitor identified in human (10) and porcine tissue (38); the gene for Kallmann syndrome in humans (39) and chicken and quail (40); WDNM1 from mouse (41) and rat metastasis (42); chelonianin isolated from the red sea turtle (Caretta caretta) egg white (43); eNAP-2, an equine anti-microbial serine protease inhibitor (44); lustrin A, isolated from the shell of the Californian red abalone (Haliotis rufescens) (45); a cortical granule protein isolated from the purple sea urchin (Strongylocentrous purpuratus) (46); and rat ps20 isolated from the urogenital sinus mesenchyme (11). (See Fig. 2 of Reference 6 for alignment of these proteins with the two domain WAPs.)

Despite sequence similarity to known serine protease inhibitors, previous reports have shown that rat WAP does not exhibit any protease inhibitory activity directed against trypsin, subtilisin, elastase, and type IV collagenase (47). Similarly, mouse WAP did not have any inhibitory activity in elastase I and II and chymotrypsin assays (48). In preliminary studies, we also have not observed any inhibition against trypsin and chymotrypsin with purified tWAP (data not shown). The protein chelonianin (IBPT), a trypsin inhibitor isolated from the egg white of red sea turtles, contains two distinctly different domains (43). The N-terminal domain contains a trypsin inhibitor domain, whereas the C-terminal domain codes for a single 4-DSC domain that inhibits subtilisin (43). It is of particular interest that the chelonianin trypsin inhibitor domain shares significant sequence identity with tammar early lactation protein, a putative trypsin inhibitor that is expressed only during phase 2A of lactation and is down-regulated around day 130 of lactation (49).

The development of a three-dimensional model for tWAP and pWAP (6) suggests the hydrophobic loops of these proteins may be functional, although the WAP family members do not share sequence identity within the active sites present in known protease inhibitors (6, 48). The major difference between the tWAP and the pWAP models arises from the fact that the "interdomain space" (between domains I and II) is only 10 residues in tWAP compared with 13 residues in pWAP and 16 residues in hSLPI (6). Furthermore, this interdomain space is conserved between domains III and I, and between I and II of tWAP (10 residues). Thus, although the tertiary fold is conserved among the WAPs, the quaternary structure of tWAP will be determined by the interdomain space. The two hSLPI domains have very little interaction with each other and appear to be capable of independent (protease inhibition) function (24). By analogy, we predict that the three tWAP domains are arranged with little interdomain interaction but with the possibility of independent function.

The surface electrostatic potential (not shown) at the top of the outer loop of each tWAP domain appears hydrophobic, analogous to that of the serine protease inhibitors. Thus, although it is unclear whether both domains of the eutherian WAPs are functional (6), all three tWAP domains have the conserved WAP (KXGXCP) motif, four intact disulfide bridges, and the surface hydrophobic character required for functionality. In particular, the high degree of structural conservation of tWAP domain II compared with hSLPI, along with its hydrophobic electrostatic character, suggests that this domain is very likely to possess protease inhibitor activity.

In eutherian species all the major milk protein genes, including the WAP gene, are induced in late pregnancy, and the proteins are secreted at unchanged levels in the milk throughout lactation (1-3, 5, 50). Mammary explant culture has been used extensively to examine the minimal hormonal requirements for the induction of milk protein genes in many eutherian species, particularly using tissue from animals in late pregnancy prior to lactogenesis. The WAP gene in mice, rats, and pigs is maximally expressed in vitro in the presence of the lactogenic hormones insulin, cortisol, and prolactin (5, 50, 51). Similarly, rabbit WAP was induced in the presence of prolactin but required the addition of insulin and cortisol for maximal expression (52). In contrast to eutherian species, the genes for beta -LG, alpha -lac, and alpha - and beta -casein are coordinately induced at parturition in the tammar (13), whereas the tWAP gene is induced around day 130 of lactation. The expression of the tWAP gene was induced in mammary explants from late pregnant tammars in response to insulin, cortisol, prolactin, tri-iodothyronine, and estradiol, but the endocrine requirement for maintenance of tWAP gene expression was limited to insulin, cortisol, and prolactin, indicating that there is a role for oestrogen and/or thyroid hormone for induction of expression of the gene. Mammary epithelial cells from phase 1 tissue are responsive to insulin, cortisol, and prolactin (13); therefore, the role of oestrogen and thyroid hormone is not to alter tissue sensitivity to these hormones. The low level of expression of the tWAP gene in tissue at day 260 of lactation was not sustained in explants cultured for up to 8 days in media with any hormone combination examined. Therefore, it appears that the inhibition of the tWAP gene observed in mammary tissue in phase 1 of lactation can be reversed in vitro and that oestrogen and/or thyroid hormone have a critical role in stimulating the transition of the mammary epithelial cells from a phase 1 to phase 2B phenotype. However, once the gene has been down-regulated in vivo, the mammary epithelial cells are no longer responsive to the combination of insulin, cortisol, prolactin, tri-iodothyronine, and estradiol to be cycled back in vitro from the phase 3 to phase 2B phenotype. The level of cortisol, prolactin, and thyroxine does not change in the peripheral circulation around days 100-130 of lactation (15), and, although the levels or insulin or estradiol have not been measured, it is unlikely that significant changes in concentration occur at this time. There is evidence to suggest that the marsupial mammary gland can alter its response to oxytocin in the peripheral endocrine milieu during development (53). Whether this mechanism extends to other hormones that regulate milk protein gene expression requires further investigation.

The secretion of tWAP only during phase 2B of lactation contrasts the secretion of this protein throughout lactation in eutherian species and provides a more viable model to examine its potential functions. It is conceivable that WAP may have multiple functions. For example, the protein is secreted as the major component of the whey fraction, therefore making it likely to be a major food source for the pouch young. During phases 2A and 2B, the physiological development of the young is largely completed (14), and the time at which secretion of tWAP is down-regulated coincides with pouch exit, a change to a mixed diet incorporating grass and milk, and the establishment of homeothermy in the young (14). Early studies (54) reported a marked increase in sulfur-containing amino acids in the milk from around day 150 of lactation, coincident with the appearance of hair follicles. Sulfur-rich amino acids are thought to be required for fur and nail growth (54), thus the secretion of the sulfur-rich WAP at this time supports their findings and suggests that it also contributes significantly to the development of pelage. Furthermore, WAP may potentially have a function in fur development in other species such as the mouse and rat, which are born with minimal fur coverage. The generation of WAP knockout mice will offer significant insights into the functions of WAP.

The expression pattern of the tWAP gene during lactation correlates with changes in expression of several other milk protein genes in the tammar, and these changes appear to be associated with specific changes in the sucking pattern of the pouch young. For example, early lactation protein is secreted only in phase 2A of lactation (49) and shares sequence identity with a trypsin inhibitor isolated from bovine colostrum (55) and a homologue secreted in possum milk (Trichosurus vulpecula (56)). The cessation of secretion of this protein occurs at the transition between phases 2A and 2B, at approximately days 100-130 of lactation and correlates with the induction of the tWAP gene. Around this time, the pouch young releases its permanent attachment to the teat but continues to remain in the pouch and suckles at intermittent intervals (14). In addition, preliminary evidence from our studies suggests that cystatin, another putative protease inhibitor is also secreted during phase 2B of lactation.4 Following the induction of tWAP, another novel protein, late lactation protein-A (LLP-A), is detected in the milk around day 170 of lactation and continues to be expressed until weaning (57). An isoform of LLP-A, late lactation protein-B (LLP-B), is induced at the transition to phase 3 at around day 220 and correlates with the down-regulation of WAP secretion. At this time the pouch young begins to exit the pouch but continues to suckle from the mother while at heel. The regulation of specific protease inhibitor genes linked to the transition between the phases of lactation raises the possibility that these proteins may contribute to either remodeling mammary tissue at this time or potentially have a role in gut development and to protect a specific dietary protein required for development of the pouch young. Earlier studies have suggested that WAP plays an important role in mammary gland development (12), because the milchlos phenotype (limited milk secretion) resulting from overexpression of the mouse WAP gene during early pregnancy in mice (48) and pigs (58) suggests that it is involved in terminal differentiation of the mammary gland. tWAP secretion declines prior to phase 3, at which time the mammary gland undergoes significant tissue remodeling, including increased growth, milk production, and secretion of a concentrated milk to provide appropriate nutrition to meet the demands of the rapidly growing young (13, 17, 18, 59). It is interesting to speculate that tWAP plays a negative regulatory role, allowing extensive mammary gland development after it has been down-regulated. Any putative role of the pouch young in regulating these changes remains to be established, but it seems likely that endocrine-stimulated tWAP gene expression during lactation is modulated by factors intrinsic to the mammary gland. This conclusion would be consistent with the independent regulation of mammary glands during asynchronous concurrent lactation (13, 15, 17).

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address and to whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. Tel.: 61-3-8344-9871; Fax: 61-3-9347-7730; E-mail: k.simpson@biochemistry.unimelb.edu.au.

Dagger Dagger Present address: Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Australian Capitol Territory 2601, Australia.

Published, JBC Papers in Press, May 3, 2000, DOI 10.1074/jbc.M002161200

2 D. Shaw, personal communication.

3 D. Shaw, unpublished data.

4 K. R. Nicholas, K. J. Simpson, and D. C. Shaw, unpublished data.

    ABBREVIATIONS

The abbreviations used are: WAP, whey acidic protein; tWAP, tammar WAP; pWAP, pig WAP; cWAP, camel WAP; mWAP, mouse WAP; platWAP, platypus WAP; 4-DSC, four-disulfide core; ALKI, antileukoproteinase; HPLC, high performance liquid chromatography; alpha -lac, alpha -lactalbumin; PAGE, polyacrylamide gel electrophoresis; beta -LG, beta -lactoglobulin; hSLPI, human secretory leukocyte proteinase inhibitor; LLP-A and -B, late lactation proteins A and B; bp, base pair(s); kb, kilobase(s); I, insulin; F, cortisol; P, prolactin; T3, tri-iodothyronine; E2, estradiol.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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