Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of a-Lactalbumin and Oleic Acid Natalia Mercer1, Boopathy Ramakrishnan1,2, Elizabeth Boeggeman1,2, Pradman K. Qasba1* 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) using polypeptide-a-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. Conclusions/Significance: We found that the molten globule form of hLA and aD-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. Citation: Mercer N, Ramakrishnan B, Boeggeman E, Qasba PK (2011) Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of a-Lactalbumin and Oleic Acid. PLoS ONE 6(10): e26093. doi:10.1371/journal.pone.0026093 Editor: Luis Eduardo Soares Netto, Instituto de Biociencias - Universidade de Sa˜o Paulo, Brazil Received May 6, 2011; Accepted September 19, 2011; Published October 10, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This project has been funded in part with Federal funds from NCI, NIH under contract HHSN261200800001E. This research was supported (in part) by the Intramural Research Program at the National Institutes of Health, National Cancer Institute, Center for Cancer Research. No external funding was received for this study. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Authors Drs. Boopathy Ramakrishnan and Elizabeth Boeggeman, who are also affiliated with the SAIC-Frederick, Inc, are funded solely by the government agency, NIH, and also have no competing interests. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing the data and materials. * E-mail: qasba@helix.nih.gov Introduction Alpha-lactalbumin (a-LA) is a 14 kDa, Ca2+-binding milk protein, synthesized in the secretory cells of lactating mammary glands. Its main function is to interact with b1,4-galactosyltrans- ferase-1 (b4Gal-T1) to form lactose synthase complex (LS). By binding to b4Gal-T1, a-LA changes the acceptor specificity of b4Gal-T1 from GlcNAc to glucose, to synthesize lactose, which is the primary carbohydrate in milk of most mammalian species [1]. Due to the similarities in gene structure and protein sequences, it has been proposed that a-LA and c-type lysozyme have evolved from the same gene [2]. As in the protein structure of c-type lysozyme, a-LA has 4 helices contained in the a-domain and b- sheets that form a b-domain. However, a-LA has a tightly bound Ca2+ in the calcium-binding loop. Removal of Ca2+ leads to a molten globule state of a-LA [3,4]. X-ray crystallographic studies on the complex of a-LA with b4Gal-T1 [5], together with enzyme kinetics studies have led to an understanding of the modulation mechanism in the LS complex [6,7]. a-LA is expressed only in mammals and in the mammary gland during lactation to function as a lactose synthase complex. However, some breast cancer cells have been found to express a-LA protein [8- 10]; a-LA has also been shown to cause apoptosis of mouse and human mammary epithelial cell lines [11] as well as of fur seal primary mammary cells, identifying it as a milk factor that regulates involution [12]. Small but detectable amounts of a-LA have been found during the early gestation phase in rat mammary gland [13]. We have also cloned the human a-LA from a cDNA library prepared from the non-lactating mammary gland that lactated previously and have used in the present studies. Thus, these studies indicate that a- LA has been at least transcribed in the breast tissues at various stages, though its function at those stages is not known. Since 1995, pioneering work by Dr. Svanborg’s group has shown that a-LA in the molten globule state complexes with oleic acid (OA), acquiring apoptotic properties toward tumor and immature cells, but not toward differentiated cells [14–16]. Extensive studies on the biological property of the complex, named HAMLET by Dr. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26093