Lysozyme and α-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula)1

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Abstract

Lysozyme and α-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14 896 and 13 985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula α-lactalbumin, along with other marsupial α-lactalbumins, formed a family distinct from the eutherian α-lactalbumins and the α-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Introduction

Although α-lactalbumin and lysozyme are functionally different, much evidence now exists that suggests that these two proteins have evolved of from a common progenitor gene 1, 2, 3, 4. Both proteins have sequence elements in common [1], a similar three-dimensional structure [4]and a similar gene structure [5]. Functionally, however, the two proteins are distinct: lysozyme has a lytic activity on bacterial cell walls whilst α-lactalbumin alters the substrate specificity of a mammary galactosyltransferase to facilitate the synthesis of the milk sugar, lactose.

Few studies have examined the structure and sequence diversity of α-lactalbumin and lysozyme from non-eutherian mammals. Two macropodid marsupial α-lactalbumin sequences have been published 6, 7but, as yet, no complete sequence for lysozyme has been reported. The sequences of two lysozyme variants from the echidna (Tachyglossus aculeatus) have been determined [8]as has the sequence of an α-lactalbumin from the platypus (Ornithorhynchus anatinus) [9], both species being monotremes.

Previously we reported the isolation and partial characterisation of α-lactalbumin from the milk of the common brush-tailed possum (Trichosurus vulpecula) [10]. The concentrations of α-lactalbumin in possum milk at different stages of lactation were also described. Here we describe the identification, purification and characterisation of lysozyme from possum milk. In addition cDNA clones for both genes have been isolated and sequenced enabling us to assess the evolutionary relationships of these two proteins.

Section snippets

Milk and tissue samples

Milk samples were obtained from captured feral possums held by Landcare Research, University of Canterbury, Christchurch, New Zealand. The pouch young were removed and the females anaesthetised with methohexitone and milked following injection of oxytocin [11]. The age of the pouch young was determined from data of Line and Verhagen [12]. Mammary glands and livers were obtained from lactating possums held by AgResearch, Invermay Research Centre, Mosgiel, New Zealand, and rats supplied by the

Identification of α-lactalbumin and lysozyme in possum milk

When analysed by 2-dimensional electrophoresis, T. vulpecula whey exhibited a relatively complex pattern compared with the whey of other species. Three proteins were apparent with Mr values of approximately 14 000 (Fig. 1). The N-terminal amino acid sequence, KDYGK, was obtained for the more acidic spot, A. This sequence matched exactly the sequence obtained previously for possum α-lactalbumin [10]. The basic spot, C, gave the N-terminal sequence, KRMERcEFARRIKQL. Based on the similarity of

Discussion

This study is the first to isolate, characterise and clone the genes for both α-lactalbumin and lysozyme from a marsupial. Previous work of Nicholas et al. [22]described the isolation of these two proteins from another possum species, Pseudocheirus peregrinus, the common ring-tail possum and reported partial amino acid sequences. When compared with the T. vulpecula sequences, there was identity at 25 out of 35 residues for α-lactalbumin and at 27 out of 49 residues for lysozyme. The T. vulpecula

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    1

    Genbank accession numbers: α-lactalbumin U34288; lysozyme, U40664.

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