Novel fusion proteins based on recombinant lectins for delivery of a cytotoxic peptide specifically to cancer cells
One of the most common post-translational modifications is covalent attachment of carbohydrates to proteins. At the cell surface, molecular recognition events involved in cancer metastasis are mediated by sugar moieties of glycoproteins. Evidence from both patient (histochemical analysis) and experimental work (tumour models) show that the metastatic tumour phenotype is associated with altered sialylation of tumour cell surfaces. Structural domains that recognize and bind specific carbohydrates without altering the recognized sugars are usually called lectins. Mistletoe lectin isoform I (MLI), from Viscum album, is an example which has strong binding affinity for sialic acids especially α2,6 sialic acid.In the present study, mistletoe lectin isoform I chain B (MLB) was targeted through conducting polymerase chain reaction (PCR) for cDNA which was reverse transcribed from the extracted V. album RNA. The targeted sequence was successfully amplified, cloned and sequenced. The sequence data showed a clear indication that the insert is our desired gene product allowing us to go on to design a fusion protein. Both N- and C- terminus fusion strategies were used to assemble constructs of the MLB chain fused to GFP in the pGFPuv expression vector which was transformed into BL21 E. coli and the expression of the target gene/fusion was successfully carried out. The same mechanism was used to generate positive and negative control fusion proteins from elderberry S. nigra lectin and snowdrop G. nivalis lectin respectively. The expressed fusions proteins were purified through affinity chromatography using CNBR-activated Sepharose 4B column coupled with degalactosylated fetuin so as to confirm the sialic acid binding activity of MLB. The binding of MLB+GFP fusion proteins to the membrane glycans of human cancer cells was also confirmed through the treatment of human metastatic melanoma cells with the fusion protein and the fusions were detected to actively attach and cross the cell membrane. Following the confirmation of MLB binding to sialic acid residues on fetuin and cancer cell membrane in which a novel attempt, the MLB sequence was further analysed by bioinformatics to identify the glycan binding domain and two main putative sugar binding sites were identified. Then a range of Lectin-Toxin fusions were designed from the whole MLB and the two glycan binding sites linked with wasp venom (Mastoparan) and honey bee venom (Melittin) through a cathepsin-B biodegradable spacer.Finally, the Lectin-Toxin fusions were screened against a range of cancer cell lines specifically WM-115 and WM 266-4 melanoma lines and a healthy melanocyte cell line (HEMn) in order to confirm the fusions binding and crossing the cell membrane of the tissues and also confirm their apoptotic effect. The fusion proteins were confirmed to have almost no cytotoxic effect on the healthy human melanocytes (HEMn). However, strong cytotoxic effect was observed during the treatment of human primary (WM-115) and metastatic (WM 266-4) melanoma cells with the fusion proteins.
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