applications of site-specific labeling to study hamlet, a tumoricidal complex of α-lactalbumin and oleic acid特定站点标签的应用研究哈姆雷特杀肿瘤的复杂α-lactalbumin和油酸.pdfVIP

Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of a-Lactalbumin and Oleic Acid 1 1,2 1,2 1 Natalia Mercer , Boopathy Ramakrishnan , Elizabeth Boeggeman , Pradman K. Qasba * 1 Structural Glycobiology Section, CCR-Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, Maryland, United States of America, 2 Basic Science Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America Abstract Background: Alpha-lactalbumin (a-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of b1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk’s main carbohydrate. When calcium is removed from a-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human a-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. Methodology/Principal Findings: In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the a-domain of hLA (aD-hLA) alone is also included in the present study. We have engineered these proteins with a 17–amino acid C-terminal extension (hLA-ext and aD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose)