Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula)

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Abstract

The whey acidic protein (WAP) is a whey protein found in the milk of a number of species. We have isolated and characterised a WAP cDNA clone from the brushtail possum (Trichosurus vulpecula) and examined its expression in the mammary gland. The amino acid sequences of WAP from the possum and another marsupial, the tammar wallaby, share 69% identity, however, less sequence identity exists between the marsupial and eutherian WAP sequences (30–37%). The possum and tammar WAP genes consist of three four-disulphide core (4-DSC) domains, with a WAP motif at the beginning of each domain. In contrast, the eutherian WAP sequences consist of two 4-DSC domains with the WAP motif only present in the second domain. This WAP motif is also present in a number of protease inhibitors found in a wide range of species. Phylogenetic analysis of marsupial and eutherian WAP sequences suggests that the ancestral WAP gene has three domains and that one of the domains has been deleted from the eutherian gene. The profile of WAP gene expression in the possum mammary gland changed throughout lactation, with WAP mRNA levels reaching a peak between days 106 and 177 of lactation. The level of WAP mRNA in the mammary gland appeared to be correlated with the level of circulating prolactin in the lactating female and was different to that observed for several other whey protein genes. Overlapping expression of the WAP and early lactation protein genes, both of which are putative protease inhibitors, may provide protection of milk immunoglobulins that are required for the prolonged period of passive immune transfer to the marsupial pouch young.

Introduction

The whey acidic protein (WAP) is present in the milk of several species including mouse [1], rat [2], camel [3], rabbit [4], pig [5] and tammar wallaby [6], but has not yet been described for primates and ruminants. Partial WAP sequences exist for the two monotremes, the platypus (Onithorhynchus anatinus; [6]) and echidna (Tachyglossus aculeatus; [6], [7]). Although the function of this protein in milk is not known, it is speculated that WAP may have a role in tissue remodelling and/or mammary gland development [8], [9]. The WAP proteins have been classed as protease inhibitors due to their homology with several families of protease and ATPase inhibitors that share a conserved domain called the ‘four-disulphide core’ (4-DSC; [10], [11]). Many of the proteins containing a 4-DSC domain bind to membrane-bound receptors and have protease inhibitor activities similar to those of the Kunitz protease inhibitors, mucous protease inhibitors, serine protease inhibitors, as well as elastase-specific and subtilisin inhibitors [6], [10], [12].

Reproduction in the Australian brushtail possum (Trichosurus vulpecula) is typified by a short gestation (17 days) followed by a prolonged period of lactation (200 days; [13]). Lactation is divided into at least three distinct phases between which milk composition changes dramatically [14], [15], [16]. Possum milk contains a large number of whey proteins most of which are homologues of eutherian milk proteins [16], [17], [18], [19]. There are, however, several other whey proteins that are not expressed in the milk of eutherian species: early lactation protein (ELP, another putative protease inhibitor [20], [21]), late lactation protein (LLP [18], [22]) and trichosurin [18]. Although most whey protein genes are expressed at similar levels throughout lactation in the possum, several genes are expressed at specific stages of lactation including ELP, LLP and transferrin [15], [16], [23], [24].

In the experiments reported here a possum WAP cDNA clone has been isolated and characterised, with the deduced amino acid sequence being analysed further. The expression profile of the WAP mRNA was established throughout lactation and was different to that observed for three other whey protein genes (ELP, LLP and transferrin) that are differentially expressed during lactation. The level of WAP mRNA was closely correlated to the level of circulating prolactin in the lactating mother.

Section snippets

Materials

The cDNA probes used have previously been described: tammar wallaby WAP ([6]), possum ELP ([20]), possum LLP ([18]), and possum transferrin [16]. The purified DNA insert for each probe was radiolabelled with [α-32P]dCTP (NEN, Boston, MA, USA) using a Rediprime random primer labelling kit (Amersham plc, Aylesbury, UK). The possum 18S rRNA probe (400 bp) was amplified by PCR and is an unpublished sequence (J. Demmer, GenBank AF089722).

cDNA cloning

Fifty thousand recombinants from the possum late lactation

cDNA cloning of possum WAP

A possum mammary gland cDNA library (late lactation) was screened with the radiolabelled tammar WAP cDNA probe. A large number of recombinants were identified in the initial screening, and the cDNA insert size was determined for 10 independent clones. The three clones with the largest cDNA inserts were analysed by DNA sequencing. The cDNA clone with the longest insert (691 bp) contained DNA sequence for the mature protein coding region and 3′ untranslated region, however, the signal peptide

Discussion

This paper reports the isolation and characterisation of a cDNA clone for possum WAP. In contrast to the high level of amino acid sequence identity between the possum and tammar wallaby WAP sequences, the sequence identity between marsupial and eutherian WAP sequences was much lower. This is consistent with the limited sequence identity observed between marsupial and eutherian milk protein genes such as β-lactoglobulin and the caseins [18], [35]. Yet other whey proteins such as α-lactalbumin

Acknowledgements

This work was supported by the New Zealand Public Good Science Fund under contract C10817, and by MAF Policy under contract PBC/09. The authors would like to thank Dr Fran Adamski for helpful discussions during this work.

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    1

    Present address: Genesis Research and Development Corporation Ltd., P.O. Box 50, Parnell, Auckland, New Zealand.

    2

    Present address: Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Vic. 3052, Australia.

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