Svanborg’s group, an acronym for human a-lactalbumin made lethal to tumor cells, have shown that it induces mitochondrial depolarization and cytochrome c release [17] and that the apoptotic response triggered by HAMLET is independent of caspase inhibition, p53 status, and Bcl-2 over expression [18]. In addition, HAMLET-induced changes are compatible with macroautophagy [19]. Other studies suggested perturbation of the proteasome structure [20] and lipid membrane integrity [21]. Besides the broad evidence of HAMLET’s anti-tumor activity, the mechanism(s) of cytotoxicity has not yet been elucidated [22]. After having first cloned a-LA and b4Gal-T1 genes [23,24] and studied their molecular interactions [5–7], here we have initiated the structure-based analysis of HAMLET using our site-specific labeling technique [25,26]. We have reproduced the previously described results by Svanborg et al., [14–16], showing that the molten globule a-LA in complex with OA kills many different tumor cells, but not untransformed or normal cells. We have designed a human a-LA (hLA) and an a-domain of hLA (aD-hLA) with a polypeptide tag at the C-terminal end, hLA-ext and aD- hLA-ext, respectively, which can be specifically glycosylated with ppGalNAc-T2, transferring a modified galactose with a chemical handle (C2-keto-galactose), as described previously [25,26], that can be further labeled with aminooxy-Alexa Fluor 488. We show here that the tumoricidal complex derived from the site-specific- labeled hLA-ext or aD-hLA-ext also kills many tumor cells and have used these labeled proteins for cell imaging. Results Protein expression and folding The recombinant hLA (124 aa) and hLA with a 17 amino acid C-terminal extension (hLA-ext) (Figure 1A) were expressed in E. coli as inclusion bodies and folded in vitro as described previously [27,28]. The near UV CD spectrum show that these proteins exist in their native state with significant tertiary structure (Figure 2A). However when the bound calcium ion is removed by dialysis against EGTA, they adopt a molten globule structure as judged by its near UV spectrum (Figure 2B). The native a-LA contains alpha (a-) and beta (b-) domains (Figure 1A, 3). The alpha-domain (83 Studies with Site-Specific Labeled HAMLET aa) comprises of the N-terminal region, residues 1 to 39, and the C-terminal region, residues 81 to 124, of the human LA. We have engineered the a-domain-form of hLA with 86 amino acids (MW 9.7 kDa) (aD-hLA), in which the beta domain has been removed and both fragments of the N- and the C-terminal region were linked by three glycine residues (Figure 1B) [29]. The a-domain hLA protein with and without the C-terminal extension were ‘‘refolded’’ in the absence of calcium salt and purified by ammonium sulfate precipitation. The near UV CD spectrum of these proteins shows that they exist in the molten globule form (Figure 2B). Although DNA sequencing of the hLA-ext and aD-hLA-ext protein genes confirmed the presence of the C-terminal extension, its presence in these proteins could be clearly seen from the SDS- PAGE gels as these proteins have a higher molecular weight compared to their respective native protein (Figures 3A and C). These gels further showed that upon the loss of the C-terminal extension peptide with thrombin protease treatment their molecular weight is found similar to their respective native protein (Figure 3A and C). Since the molecular weight of the released C- terminal polypeptide is only 1337 Da it could not be observed on any SDS-PAGE gel, therefore, a MALDI-TOF spectroscopic analysis of these thrombin treated samples was carried out. Such analysis shows a release of correct molecular weight peptide upon the treatment of thrombin from these proteins (Figure 3B), confirming the presence of thrombin cleavable C-terminal extension peptide in these proteins. Site-specific labeling of hLA-ext and aD-hLA-ext protein molecules Using the ppGalNAc-T2 enzyme, C2-keto-galactose from UDP-C2-keto-Gal was transferred to the single Thr residue, located in the polypeptide extension of the native hLA-ext protein (Figure 4A & B). MS analysis of the thrombin cleaved peptide from the glycosylated hLA-ext protein showed an increase in molecular mass by 201 Da that corresponds to a single C2-keto-gal sugar moiety, thus confirming the presence of a single sugar moiety in the C-terminal extension peptide (Figure 4B). The glycosylated Figure 1. Schematic diagram of the hLA and aD-hLA proteins with C-terminal extension. (A) hLA (protein sequence acc. No J00270) and hLA-ext proteins with the crystal structure of hLA (far right) (pdb 1a4v) showing alpha and beta domains. (B) aD-hLA and aD-hLA-ext proteins with the corresponding alpha domain structure (model) (far right). Since the alpha domain is comprised of N-terminal residues, 1 to 39, and the C-terminal residues, 81 to 124, of hLA (cyan colored), a three residue glycine linker was used to fuse the N-terminal and C-terminal fragments to construct the alpha domain form of the protein (aD-hLA) where beta domain is removed. A 17-amino-acid extension containing a thrombin cleavage site and a Thr residue (substrate for ppGalNAcT2 enzyme), was engineered at the C-terminal domain of hLA (A, hLA-ext) and aD-hLA (B, aD-hLA-ext). doi:10.1371/journal.pone.0026093.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26093