Monomer/dimer equilibrium of the AB-type lectin from mistletoe enables combination of toxin/agglutinin activities in one protein: analysis of native and citraconylated proteins by ultracentrifugation/gel filtration and cell biological consequences of dimer destabilization.
The biological activity of a lectin is influenced by its quaternary structure. Viscumin is special among the family members of toxic AB-type plant lectins, because it triggers mitogenicity, toxicity, and agglutination. Its activity profile is dependent on the concentration, motivating a thorough inspection of the status of quaternary structure. Over a broad range of protein concentrations (0.01-25 mg/mL), viscumin occurs as a dimer. At high concentrations, the solutions exhibited nonideality, self-association, and polydispersity in sedimentation equilibrium and velocity experiments caused by irreversible aggregation. Calculation of viscumin's overall shape based on sedimentation velocity data resulted in an elongated dimer form resembling that of crystallized agglutinin. Appearance of monomers was restricted to concentrations in the submicrogram/mL level, as demonstrated by fast protein liquid chromatography gel-filtration analysis. To shift the equilibrium to the monomer for comparative cell biological assays, we performed chemical modification under conditions protecting the lectin activity. Citraconylation was effective to destabilize the dimer. Binding studies by fluorescence-activated cell scan analysis revealed a reduction in cell association upon modification and a tendency for increased sensitivity towards haptenic inhibitors at microg/mL concentrations. Nonetheless, growth inhibition continued to be potent for the ricin-like monomer despite reduced extent of binding. Occurrence of a concentration-dependent monomer/dimer equilibrium appears to achieve the same objectives as the development of two separate protein entities in Ricinus communis, an alternative strategy to emergence of a monomeric toxin, and cell cross-linking dimeric agglutinin.
Jiménez
Marta
M
Instituto de Química Fisica Rocasolano, CSIC, 28006 Madrid, Spain.
Sáiz
José L
JL
André
Sabine
S
Gabius
Hans-J
HJ
Solís
Dolores
D
D000373:Agglutinins / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002850:Chromatography, Gel; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D037102:Lectins / Q000737:chemistry; D008207:Lymphatic Metastasis; D046911:Macromolecular Substances / Q000737:chemistry; D051379:Mice; D014784:Mistletoe; D008968:Molecular Conformation; D041681:NIH 3T3 Cells; D028321:Plant Preparations / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D054790:Ribosome Inactivating Proteins, Type 2; D012278:Ricinus; D013997:Time Factors; D014118:Toxins, Biological / Q000737:chemistry*; D014461:Ultracentrifugation
D000373:Agglutinins; D037102:Lectins; D046911:Macromolecular Substances; D028321:Plant Preparations; D010940:Plant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C488862:mistletoe lectin I; C009674:ribosome inactivating protein, Viscum