title: Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.
abstract: Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular 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 the 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 the enzymatic activities of the type II RIP A-chains.
authors.affiliation: BRAIN GmbH, Zwingenberg, Germany. je@brain-biotech.de
authors.forename: J
authors.initials: J
authors.lastname: Eck
authors.forename: M
authors.initials: M
authors.lastname: Langer
authors.forename: B
authors.initials: B
authors.lastname: Möckel
authors.forename: A
authors.initials: A
authors.lastname: Baur
authors.forename: M
authors.initials: M
authors.lastname: Rothe
authors.forename: H
authors.initials: H
authors.lastname: Zinke
authors.forename: H
authors.initials: H
authors.lastname: Lentzen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D007525:Isoelectric Focusing; D037102:Lectins / Q000737:chemistry / Q000235:genetics*; D014784:Mistletoe / Q000235:genetics*; D008969:Molecular Sequence Data; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D010946:Plants, Medicinal; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological / Q000737:chemistry / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018744:DNA, Plant; D037102:Lectins; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum