Evolution of Lactose and Milk Oligosaccharides
	Mammalian milk or colostrum contains a few percent carbohydrates, of which disaccharide lactose (Gal(1-4)Glc) usually makes up more than 80%. Free lactose is synthesized within lactating mammary glands from UDP-galactose (donor) and glucose (acceptor) by a transgalactosylation catalyzed by a complex of a 4galactosyltransferase I (4GalT I) and -lactalbumin (-LA), one of the milk proteins. As other tissues do not contain -LA but do contain 4GalT I, the expression of -LA within the mammary gland is the key to the presence of lactose in milk. Although the amino acid sequence and tertiary structure of -LA are similar to those of c – type lysozyme (Lz), which cleaves the bond in peptidoglycans of bacterial cell walls, the usual -LA has lost two residues of Glu-35 and Asp-53, which are essential for the catalytic activity of Lz. However, the -LA of Australian monotremes, platypus and echidna, whose unique characteristic is their oviparous mode of reproduction, had Glu-35 as well as Asn-44, Ala-109 and Trp-113, which are almost always found in Lz. Because it is thought that -LA, a mammalian specific protein, has occurred through the evolution of Lz, the monotreme -LA can be thought of as proteins which have undergone relatively little change compared with other -LA during evolution. From the above, it is thought that mutation has occurred in the non-essential positions for the catalytic acitivity in Lz to produce the bifunctional protein, then the one catalytic position of Lz, Asp-53, has been lost as found in the extant monotreme -LA, and then another catalytic position, Glu-35, has also been lost as in other -LA during the course of evolution (Fig. 1).
Fig.1. Molecuar evolution from lysozyme to -lactalbumin
	Mammalian milk or colostrum usually contains less than 20% of milk oligosaccharides, which consist of many saccharides with a lactose unit in the reducing end of the carbohydrate fraction, too, as well as lactose, while the milk of monotremes, marsupials, and bears, a eutherian species, contains more milk oligosaccharides than lactose. The heterogeneity of them has been found among the mammalian species. Human milk contains more than 100 oligosaccharides whose core units are classified into 12 groups, e.g., lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexaose (LNH) and lacto-N-neohexaose (LNnH), etc. One hundred oligosaccharides are built by the attachment of Lewis a, b, x or 2-3/2-6 Neu5Ac to the twelve core units. In milk of most platypus the higher oligosaccharides have a LNnT or a LNnH core unit with Lewis x or Lewis y. The major series of milk oligosaccharides of tammar wallaby, a marsupial, has one to five residues of (1-3)- linked Gal attached to the reducing lactose unit. In most bear milk the higher oligosaccharides have an LNnT or an LNnH core unit with Lewis x and ABH antigens or an -Gal epitope. In terms of chemical structure, eutherian and monotreme oligosaccharides resemble each other whereas the marsupial oligosaccharides are mostly unique. When the young of eutherians including human consume mother’s milk, the lactose is split into galactose and glucose by intestinal lactase, which is located in the membrane of the microvilli of the brush border of the small intestine, and the monosaccharides are transported into the enterocytes by a specific mechanism. The monosaccharides are used as energy sources in the young. The intestinal lactase is completely absent from the brush border of the villi of the small intestine of the suckling tammar wallaby. On the other hand, it is suggested that most milk oligosaccharides are not absorbed in human young, while they are regarded to be anti - infection factors to inhibit the attachment of pathogenic bacteria and virus in the colon. It is assumed that the milk oligosaccharides are absorbed in the small intestine of marsupial or monotreme young by pinocytosis or endocytosis and are then hydrolyzed by lysosomal glycosidases. It is thought that they are used as an energy source in the young and also act as anti-infection factors for them. Based on the above, the following hypothesis is proposed for the evolution of lactose and milk oligosaccharides. The proto-lacteal secretions of the primitive mammary glands of the common ancestor of mammals contained fat and protein but not lactose and milk oligosaccharides because of non-expression of -LA. When -LA first appeared, its content within the lactating mammary glands was low and lactose was synthesized at a relatively slow rate. Because of the presence of glycosyltransferases, almost all of the lactose was utilized for the synthesis of oligosaccharides. The predominant saccharides in the proto-lacteal secretions or milk produced by this common ancestor were oligosaccharides and not free lactose. Initially, the oligosaccharides served mainly as anti-infection factors against pathogenic organisms along with other defense factors. They were then recruited as an energy source for the neonates; this was achieved by an increase in the synthesis of -LA and possibly also of the glycosyltransferases. The two biological roles of anti-infection and provision of energy were preserved in both monotremes and marsupials but in most eutherians the milk concentration of free lactose increased due to a significant increase in -LA synthesis by the mammary gland. Lactose therefore became a significant energy source for most eutherians while oligosaccharides continued to serve mainly as anti-microbial agents. The advent of milk lactose at relatively high concentrations necessitated the prior evolution or co-evolution of brush border small intestinal lactase, which provided an efficient mechanism for the digestion of lactose.
	Tadasu Urashima (Obihiro University of Agriculture and Veterinary Medicine)
	References (1) Urashima T, Saito T, Nakamura T, Messer M: Oligosaccharides of milk and colostrum in non-human mammals. Glycoconjugate J. 18, 357-371, 2001 (2) Messer M, Urashima T: Evolution of milk oligosaccharides and lactose. Trends Glycosci. Glycotechnol. 14, 153-176, 2002 Links GP-B03 A -1,4-Galactosyltransferase Family (Takeshi Sato)
Jul. 21, 2004