Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain

Mistletoe lectin I (MLI) is the major active constituent of mistletoe extra cts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodi meric disulphide-linked glycoprotein is classified as type II ribosomein-ac tivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-su bunit, also referred to as toxic entity. MLI and the close relative ricin b oth belong to the family of the two-chain plant type II RIP proteins. Isola tion of the glycosylated proteins from plant material yield inhomogeneous m aterial probably due to posttranslational modifications. The aim of this st udy was to prepare pure and homogeneous protein as a prerequisite for struc tural and mechanistic studies in order to gain insight into the mode of act ion of this cytotoxic plant protein on tumour and immune cells. Of particul ar interest was to explain whether the differences in toxicity of ML and ri cin are the result of variations of their enzymatic activities. By investig ating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific ampl ification of the mistletoe lectin gene. Applying this PCR strategy the full -length 1923 nucleotide DNA sequence coding for the prepro-protein was obta ined showing the existence of a single intron-free gene. In order to elucid ate the molecular basis for the observed differences in cytotoxicity within the family of RTP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coil BL21/pT7 resulted in productio n of insoluble inclusion bodies constituting 20-30% of total protein. Refol ding led to a pure and homogeneous protein species with an apparent molecul ar mass of 27 kDa and a pi value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in th e same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in t he enzymatic activities of the type II RIP A-chains.