Alpha-lactalbumin affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase: synthesis of GalNAc beta 1-->4Glc.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.

Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.

Foldons, protein structural modules, and exons.

Foldons, which are kinetically competent, quasi-independently folding units of a protein, may be defined using energy landscape analysis. Foldons can be identified by maxima in a scan of the ratio of a contiguous segment's energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. The predicted foldons are compared with the exons and structural modules for 16 of the 30 proteins studied. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules, but there are marked sequence-dependent effects. There is only a weak correlation of foldons to exons. For gammaII-crystallin, myoglobin, barnase, alpha-lactalbumin, and cytochrome c the foldons and some noncontiguous clusters of foldons compare well with intermediates observed in experiment.

Apoptosis induced by a human milk protein.

To the breast-fed infant, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. While investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state--multimeric alpha-lact-albumin--is a potent Ca(2+)-elevating and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric alpha-lactalbumin killed all transformed, embryonic, and lymphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by furnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric alpha-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.