**2. Origin of HAMLET - Human α-lactalbumin made lethal for tumor cells**

Complexes of human α-lactalbumin with OA were discovered by Catharina Svanborg and co-workers about two decades ago [5]. Initially, Håkansson et al. [5,6] and Svensson et al. [7] discovered that a multimeric human -lactalbumin derivative isolated from the casein fraction of milk was a potent Ca2+-elevating and apoptosis-inducing agent with a broad, yet selective cytotoxic activity. It was found that the apoptosis-inducing fraction of lactalbumin contained oligomers of -lactalbumin that have undergone a conformational change towards a molten globule-like state [7]. Oligomerization appeared to have conserved

© 2012 Vukojević and Morozova-Roche, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Similar cytotoxic activity was observed by in vitro produced HAMLET complexes, in which human -lactalbumin was converted into the apoptosis-inducing tumoricidal folding variant by binding OA [8-10]. The formation of HAMLET was carried out in chromatography ion exchange columns preconditioned with fatty acids. It was also identified that HAMLET formation is governed by stereo-specific lipid-protein interactions and that only unsaturated C16-C20 fatty acids in cis conformation, but not other fatty acids could induce HAMLET [11]. Among such complexes, only HAMLET complex with OA and cis vaccenic acid complexes were shown to kill tumor cells efficiently, while the C16 or C20 cis fatty acid complexes with -lactalbumin showed low or intermediate activity [11].

HAMLET's remarkable tumor-selective cytotoxicity correlated with the conformational change of the protein that has taken place upon complex formation, i.e. conversion to molten globule-like state. However, α-lactalbumin in a molten globule state without OA does not possess such activity per se, indicating that the presence of both components is required. As partially unfolded α-lactalbumin can revert easily to its native state upon Ca2+ binding in natural cell culture media or within cells, the D87A Ca2+- binding site mutant of α-lactalbumin was produced [12], which was lacking Ca2+-binding property and remained partially unfolded at physiological conditions. Such mutant formed a tumoricidal HAMLET-like complex with OA, but the partially unfolded protein alone did not kill tumor cells. Another non-native -lactalbumin variant with all amino acids building disulfide bridges substituted by Ala residues also did not exhibit cytotoxic activity in the absence of OA, while its HAMLET-like form displayed strong tumoricidal activity against lymphoma and carcinoma cell lines [13]. Together, these experiments consistently confirmed that both molten globule like protein conformation and specific fatty acids are required for the tumoricidal activity of the investigated complexes.

It has been suggested that naturally occurring HAMLET may have a protective function. In the stomach of nursing children low pH can induce the release of Ca2+ from the high-affinity Ca2+-binding site of α-lactalbumin and activate lipases hydrolyzing free fatty acids from milk triglycerides, thereby providing naturally occurring conditions that favor the formation of -lactalbumin lethal to tumors [14]. This could be important for lowering the incidence of cancer in breast-fed children by purging tumor cells from the gut of the neonate.

### **3. Equine lysozyme (EL) as a structural homologue of α-lactalbumin**

664 Lipoproteins – Role in Health and Diseases


lactalbumin but nuclear uptake of multimeric -lactalbumin is independent of Ca2+.

cis fatty acid complexes with -lactalbumin showed low or intermediate activity [11].

tumoricidal activity of the investigated complexes.

HAMLET's remarkable tumor-selective cytotoxicity correlated with the conformational change of the protein that has taken place upon complex formation, i.e. conversion to molten globule-like state. However, α-lactalbumin in a molten globule state without OA does not possess such activity per se, indicating that the presence of both components is required. As partially unfolded α-lactalbumin can revert easily to its native state upon Ca2+ binding in natural cell culture media or within cells, the D87A Ca2+- binding site mutant of α-lactalbumin was produced [12], which was lacking Ca2+-binding property and remained partially unfolded at physiological conditions. Such mutant formed a tumoricidal HAMLET-like complex with OA, but the partially unfolded protein alone did not kill tumor cells. Another non-native -lactalbumin variant with all amino acids building disulfide bridges substituted by Ala residues also did not exhibit cytotoxic activity in the absence of OA, while its HAMLET-like form displayed strong tumoricidal activity against lymphoma and carcinoma cell lines [13]. Together, these experiments consistently confirmed that both molten globule like protein conformation and specific fatty acids are required for the

It has been suggested that naturally occurring HAMLET may have a protective function. In the stomach of nursing children low pH can induce the release of Ca2+ from the high-affinity Ca2+-binding site of α-lactalbumin and activate lipases hydrolyzing free fatty acids from milk triglycerides, thereby providing naturally occurring conditions that favor the formation of -lactalbumin lethal to tumors [14]. This could be important for lowering the incidence of

cancer in breast-fed children by purging tumor cells from the gut of the neonate.

Similar cytotoxic activity was observed by in vitro produced HAMLET complexes, in which human -lactalbumin was converted into the apoptosis-inducing tumoricidal folding variant by binding OA [8-10]. The formation of HAMLET was carried out in chromatography ion exchange columns preconditioned with fatty acids. It was also identified that HAMLET formation is governed by stereo-specific lipid-protein interactions and that only unsaturated C16-C20 fatty acids in cis conformation, but not other fatty acids could induce HAMLET [11]. Among such complexes, only HAMLET complex with OA and cis vaccenic acid complexes were shown to kill tumor cells efficiently, while the C16 or C20 The protein component of ELOA is equine lysozyme (EL), a protein that is abundant in mare milk and kumys (a fermented beverage produced from mare milk that is widely used in Middle Asia). EL belongs to an important calcium-binding sub-family within the extended family of lysozymes, *i.e.* in contrast to common c-type lysozyme EL possesses high affinity calcium binding site, resembling with this regards α-lactalbumins. Lysozymes and lactalbumins are characterized by not more than 35-40% in sequence homology, but share remarkably similar tertiary folds. EL serves as an evolutionary bridge between lysozymes and -lactalbumins, combining the structural and folding properties of both. These are rather small molecules of about 14.6 kDa, consisting of two sub-domains – -helical and sheet rich domains separated by a deep cleft. Lysozyme active site is located in this cleft (absent in α-lactalbumins). Calcium is coordinated by a loop positioned at the bottom of the cleft and important for the structural integrity of the protein, yet the physiological function of calcium binding to EL and other calcium-binding lysozymes is still unclear. The calciumbinding usually increases the protein stability against denaturing treatments, however in the case of EL, the significantly lower stability and cooperatively was observed compared to non-calcium-binding lysozymes even in its holo-form, while in the apo-form its thermodynamic stability is closer of -lactalbumins than to c-type lysozymes [15,16]. EL forms a wide range of partially folded states under equilibrium conditions similar to these of -lactalbumins [16,3,17,18]. However, EL molten globule is much more structured compared to the "classical" molten globules of α-lactalbumins, possessing an extended native-like hydrophobic core stabilised by interactions between three major α-helices (A, B and D-helices) in the α-domain [17,18]. Like c-type lysozymes, during refolding kinetics EL forms an ensemble of well-defined transient kinetic intermediates, possessing very persistent structures [19]. Importantly, the rapidly formed kinetic intermediate of EL (2.5 ms refolding time) is characterised by the same extended core structure as its equilibrium molten globule analogues populated under acidic conditions, indicating that the hydrophobic collapse into molten globule-like state is an essential step in protein folding. Given its distinct structural properties, EL may be used as an invaluable research object in revealing the general mechanism and role of intermediate states in protein folding.

### **4. Controlled ELOA production using ion-exchange chromatography**

Similar to HAMLET, ELOA was produced at the solid-liquid interface in an ion-exchange chromatography column preconditioned with OA (Figure 1).

ELOA was eluted as a strong peak by using a 0-1.5 M NaCl gradient. In the absence of OA, free EL was eluted as a narrow peak at a low NaCl concentration [4]. EL was subjected to column chromatography without decalcification as it has been performed with human lactalbumin during original HAMLET production, indicating a difference in the generic properties of EL and -lactalbumin. ELOA complex remains stable in its lyophilized form suitable for long storage as well as it can be kept in solution for up to a week. It is also important to note, that co-incubation of a 50 fold excess of OA mixed with EL in solution at room temperature did not lead to ELOA formation as was evident from the lack of characteristic ELOA conformational transitions monitored by near-UV CD [4]. Thus, the application of a solid-liquid interface facilitating protein self-assembly and protein-OA interactions proved to be an efficient approach in production of both ELOA and HAMLET complexes. By comparison, the complex of hen egg white lysozyme with oleic acid was also produced under the same conditions, but it was very low populated, unstable and OA can be easily depleted from its structure. Hen egg white lysozyme is much more stable than EL and it is evident that the hydrophobic interface in the column chromatography is not sufficient to cause its partial unfolding and interactions with OA molecules.

**Figure 1. Schematic presentation of ELOA formation at the solid–liquid interface in a Sepharose chromatography column.** The positively charged Sepharose matrix is preloaded with oleic acid (the hydrophilic carboxyl group is denoted by a blue circle and the aliphatic chain by a gray line with a "kink" at the position of the double bond). When folded, EL molecules (shown in space-filling representation, with exposed hydrophilic residues in purple and buried hydrophobic residues in grey) are added to the column some hydrophobic residues become exposed and interact with oleic acid molecules forming ELOA.

Indeed, hydrophobic and charged surfaces often facilitate the self-assembly processes by recruiting proteins and modifying their interactions [20]. Within the ion-exchange matrix bound OA molecules constitute an extended surface, facilitating both charged and hydrophobic interactions with EL molecules, while in solution OA, like many other small aliphatic molecules, would be present as a micelle. In addition, the solid-liquid interface may induce EL partial unfolding and expose its hydrophobic surfaces buried in the native state; this can also be critical for ELOA complex formation. It is worth noting, that hydrophobic interactions within the column chromatography may effectively model the interactions, which can take place at the hydrophobic and charged surfaces in biological systems. For example, the interactions with cell lipid membranes may be able to induce protein-ligand complexation otherwise not occurring in solution.
