Complete Structure Determination Of N-Acetyl-Dgalactosamine-Binding Mistletoe Lectin-3 From Viscum Album L. Album

The primary structure of the B chain of the N-acetyl-D-galactosamine-recognizing mistletoe lectin-3 (ML-313) 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-313 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 Asn(95) and Asn(135) 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 RlPs, although there are remarkable differences in the D-galactose-specific mistletoe lectin-1B chain. The recently published primary structure of the mistletoe lectin-3A chain [1] and the now available primary sequence of the 3A 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 (c) 2004 European Peptide Society and John Wiley & Sons, Ltd.