Abstract
HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.
Highlights
HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro, including carcinoma and lymphoma cells from different species and has shown therapeutic efficacy against glioblastomas, papillomas and bladder cancer in vivo [1,2,3]
Small angle X-ray scattering (SAXS) patterns of HAMLET were recorded as described in Materials and Methods to yield the final composite scattering curve shown in figure 1A, which indicates a monodispersed protein in solution
Comparison of the forward scattering of HAMLET with the values obtained from a reference solution of bovine serum albumin, (BSA; 66.462 kDa) yields a molecular mass of 1562 kDa, in agreement with the mass of a-lactalbumin (24), indicating that HAMLET is monomeric at the concentrations used
Summary
HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro, including carcinoma and lymphoma cells from different species and has shown therapeutic efficacy against glioblastomas, papillomas and bladder cancer in vivo [1,2,3]. While a-lactalbumin acts as the glucose specifier for b-1,4-galactosyltransferase [15,16], the native protein does not form tumoricidal complexes with oleic acid, demonstrating that partial unfolding of the protein alters its activity. Based on these findings, we have proposed that proteins may respond to different environments by changing their fold and binding partners and that this process allows a single polypeptide chain to exert vastly different biologic functions in different tissue compartments [11]
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