Complete structure determination of the A chain of mistletoe lectin III fromViscum album L. ssp.album (original) (raw)
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Archives of Biochemistry and Biophysics, 2004
Ribosome-inactivating proteins having antitumor and immunomodulatory properties constitute the active principle of widely used mistletoe therapy in Europe. This is the first report of the four isoforms of Himalayan mistletoe ribosome-inactivating proteins (HmRips) from Viscum album parasitized on wild apple inhabiting NW Himalayas. HmRips were purified by affinity chromatography and four isoforms were separated by ion-exchange chromatography. HmRip 1, 2, 3, and 4 have isoelectric points of 6.6, 6.1, 5.2, and 4.7, respectively. Disulfide linked toxin and lectin subunits of HmRip 1 and 2 isoforms have molecular weights of 28 and 34 kDa while those of HmRip 3 and 4 have 28 and 32 kDa. The isoforms lacked blood group specificity and showed positive activity with seven mammalian erythrocyte types but did not show any activity with avian erythrocyte type. Lectin activity of HmRips remained unchanged for a wide range of temperatures (0–65 °C) and pH (3–9). Unlike other type II Rips, the HmRip 1, 2, and 4 showed unique affinity towards l-rhamnose, meso-inositol, and l-arabinose while HmRip 3 has specificity to gal/galNAc. Sugar binding studies with 22 sugars also suggested that the C-4 hydroxyl of galactose might be the critical site involved in sugar binding of HmRips. Type II Rips are known to be galactoside specific and do not have affinity for l-rhamnose and meso-inositol. However, HmRip 1, 2, and 4 having equal affinity for galactose and l-rhamnose do not strictly fit into any of the four structural classes of the lectins and represent a new class of type II Rips and plant lectins.
Journal of Peptide Science, 2005
The primary structure of the B chain of the N-acetyl-D-galactosamine-recognizing mistletoe lectin-3 (ML-3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC-separation and Edman degradation and confirmation of the peptide sequences by MALDI-MS. ML-3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N-glycosylation sites at positions Asn95 and Asn135 of the lectin, were determined by a combination of glycosidase treatment and MALDI-MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type-II RIPs, although there are remarkable differences in the D-galactose-specific mistletoe lectin-1B chain. The recently published primary structure of the mistletoe lectin-3A chain1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML-3. The model demonstrates, unequivocally, that ML-3 is a member of the type-II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML-1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin-3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.
Journal of biochemistry and molecular biology, 2006
Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to ...