Monotreme whey proteins: isolation, characterization and comparative aspects

Abstract

Lactation is a specific mammalian adaptation. Milk is the principal source of nutrition for the newborn mammal. Both its needs and the physiology of lactation vary among the different mammalian species. These factors are reflected in the differing milk compositions, in particular the protein compositions. Thus, comparative studies of the protein composition of eutherian, metatherian and prototherian mammals are of great importance in their own right. Furthermore, these proteins exhibit an extraordinary array of conformations and protein-protein interactions. Thus, their study will also throw light on basic aspects of protein structure and physiological function. This thesis is concerned with a study of the milk proteins of the egg-laying mammals (monotremes), the echidna and platypus. At the time of commencement of this study little was known of the protein composition of their milk. Thus, it was necessary to establish initially the overall protein composition of the limited number and volume of milk samples available. This was achieved by electrophoretic and immunological studies, fractionation of the proteins and determination of certain properties, e.g. molecular weight, amino acid composition and partial amino acid sequence. Efforts were concentrated on the whey proteins and where possible they were identified in terms of known proteins, the bovine whey proteins being used as a point of reference. In addition, two groups of whey proteins were chosen for more intensive study: the lysozymes and iron (III) binding proteins. The main echidna whey proteins have been resolved into eleven bands on starch gel electrophoresis at pH 7.G. These were designated A through K on the basis of decreasing electrophoretic mobility. Nine bands, designated 1 through 9, have been resolved in a sodium dodecyl sulphate-gradient polyacrylamide gel system. The relationship between the bands of the two gel systems has been determined and proteins corresponding to Bands A, B, C, E, G-J have been isolated. Band A protein has an apparent molecular weight of ca. 17,800 and appears to have no counterpart in echidna blood serum. Twenty-five residues of the TV-terminal sequence have been determined. It bears no simple relationship to any known protein, but has ca. 30% homology with an acidic whey protein precursor found in rat and mouse milk. Band B protein has an apparent molecular weight of ca. 55,000 and a counterpart is not detected in the blood serum. The sequence of the first fifteen TV-terminal residues was determined. Sequence homology with any known protein is not evident. Band C protein occurs in the milk and blood and is echidna serum albumin. Both milk and blood protein have the. same electrophoretic mobility and apparent molecular weight. The determination of the TV-terminal sequence indicates 63% homology with human and bovine serum albumin over the first 30 residues. Band E protein occurs only in the milk, and has an apparent molecular weight of ca. 21,000. The sequence of the first 68 TV-terminal residues indicates that it is a novel protein. Bands G-J and a minor band (G ) evident in some milk samples are echidna transferrins (milk). The electrophoretic patterns vary depending on the sample, but all exhibit 3-4 of the bands. Bands H-J are evident in the electrophoretic patterns of the blood serum. The milk bands are identified as transferrins on the basis of their electrophoretic mobilities, ,r,^Fe(III) binding capacity, molecular size and A-terminal sequences. The multiple banding reflects differences in the numbers of sialic acid residues (0-4) present. The principal cathodic electrophoretic band in the milk is lysozyme. Echidna milk is relatively rich in this protein, containing ca. 2 mg mf*. Previous observations that there is a lysozyme, echidna lysozyme 1, occurring'in the milk of Tachyglossus aculeatus rnultiaculeatus and another, echidna lysozyme II, of different electrophoretic mobility at pH 5.3, occurring in Tachyglossus aculeatus aculeatus, have been confirmed. Each enzyme has been isolated and the complete amino acid sequences determined. They are both c-type lysozymes, M 13,988 and 13,861 respectively. They differ in three amino acid residues. Both proteins have a number of unusual features, e.g. some residues considered important in a-lactalbumins for lactose synthase activity are conserved. However, contrary to previous findings in this laboratory, echidna lysozyme I isolated during the course of the present study, does not appear to be active in the lactose synthase system. Furthermore, evidence is obtained for the presence of very low concentrations of a conventional o-lactalbumin. Proposals for further work on this particular problem are presented. Seven electrophoretic bands are observed on starch gel electrophoresis (pH 7.6) of platypus whey proteins. These are designated A through G. Their apparent molecular weight range from 19.000-79,000. Proteins corresponding to Bands A, C, D, F and G have been isolated and preliminary characterization made. Bands F and G proteins are shown to be transferrins. The major blood serum iron (III) binding protein has a similar electrophoretic mobility to Band G of the milk. Platypus arid echidna transferrins exhibit 80% homology over the first 30 A-terminal residues. Only trace levels of lysozyme activity and weak lactose synthase activity were detected in the 18 platypus milk samples examined. The significance of the work in the overall framework of comparative studies of milk proteins is discussed.