RESUMEN
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 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 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.
Asunto(s)
Acetilgalactosamina/química , Preparaciones de Plantas/química , Preparaciones de Plantas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Toxinas Biológicas/química , Toxinas Biológicas/aislamiento & purificación , Alquilación , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía de Afinidad , Secuencia Conservada , Cisteína , Electroforesis en Gel de Poliacrilamida , Pruebas de Hemaglutinación , Humanos , Espectrometría de Masas , Muérdago , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Inactivadoras de Ribosomas , Proteínas Inactivadoras de Ribosomas Tipo 2 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A method was established to isolate and quantify small amounts of chitin-binding mistletoe lectin (cbML) from extracts of the mistletoe (Viscum album L.) by affinity and reverse phase high performance liquid chromatography. A validation, according to ICH guidelines, of this analytical method was carried out and showed that specificity, robustness and precision are guaranteed. In addition, linearity is ensured for a content between 0.6 and 4.1 microg/ml of cbML in the extracts and recovery was calculated to be in the range of 94 to 100%. So, accuracy of the method is guaranteed as well. As far as the range of the analytical method is concerned, a minimum of 1.2 microg and a maximum of 8.2 microg cbML can be incubated with the affinity material. Detection and quantitation limits were calculated to be 0.13 and 0.46 microg/ml cbML, respectively.
Asunto(s)
Lectinas/química , Muérdago/química , Secuencia de Aminoácidos , Calibración , Quitina/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , Extractos Vegetales/química , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.