alpha -Lactalbumin (LA) Stimulates Milk beta-1,4-Galactosyltransferase I (beta 4Gal-T1) to Transfer Glucose from UDP-glucose to N-Acetylglucosamine: CRYSTAL STRUCTURE OF beta 4Gal-T1·LA COMPLEX WITH UDP-Glc*

beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha -lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the Km for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the Km for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1·LA complex with UDP-Glc·Mn2+ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed

A Chemoenzymatic Approach toward the Rapid and Sensitive Detection of O-GlcNAc Posttranslational Modifications

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of β-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications

Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid

umor cells), and its tumoricidal activity has been well established.-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules.We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity

DNA and Gene Therapy: Transfer of Mouse DNA to Human and Mouse Embryonic Cells by Polyoma Pseudovirions

It was demonstrated previously that polyoma pseudovirions (DNA fragments encapsidated by polyoma virus coats) are adsorbed to and uncoated by mouse-embryo cells. 24 hr after infection of secondary mouse-embryo cells with [(3)H]thymidine-labeled pseudovirus, 24% of the total radioactivity found in cells occurred in the nuclear fraction. This nuclear radioactivity represents pseudovirus DNA, as shown by its hybridization with mouse-embryo DNA. Since much of the radioactive material found in the nuclear fraction is sensitive to pancreatic DNase, it is available as uncoated DNA. 24 hr after infection of human embryo cells with [(3)H]thymidine-labeled pseudovirions, 7% of the total cellular radioactivity is found in the nuclear fraction of the cells. That this radioactivity represents uncoated pseudoviral DNA was shown by sedimentation of the disrupted nuclear fraction through a neutral sucrose gradient. The pseudoviral DNA in the nuclear fraction has been nicked to some extent, as shown by the heterogeneity of the peaks found after sedimentation of the disrupted nuclear fraction through alkaline sucrose. The experiments demonstrate that polyoma pseudovirions deliver DNA to human cells