1. Lectin leftovers
2/27/07

2. Lectin leftovers Brief review of animal lectins
Brief overview of plant lectins
Determining/detecting lectin activities

3. Lectins
Carbohydrate binding proteins that are not antibodies or enzymes
Bind with high specificity
Latin: lectus, meaning to gather or select
Relatively high dissociation constants (ca 100 M)
Carbohydrate recognition domains are small
Most lectins are multivalent
After Alvarez-Manilla

4. Animal lectins are everywhere
After Schnaar, R.L.

5. After Schnaar, R.L.

6. Common structural features of animal lectins
After Schnaar, R.L.

7. Lectins are present in all organisms
Virus----- Influenza
Bacteria binding to hosts during pathogenesis
Vegetable
Many have been purified and characterized
Physiological function is unknown
Animal
Several proteins with a wide variety of functions
After Alvarez-Manilla

8. Vegetable Lectins Leguminosae Graminae Solanaceae
ConA (Concanavalin A from Jack bean)
Phaseolus Vulgaris (PHA-L and PHE)
Soy bean agglutinin
Graminae
Wheat germ agglutinin
Solanaceae
Tomato lectin
Potato lectin
After Alvarez-Manilla

9. Structure of Vegetable lectins
Compact -barrel, no alpha helices
Antiparalell beta-sheets
Many require metals (leguminosae)
Ca and Mn
Metals do not participate directly in the binding but are required
After Alvarez-Manilla

10. Structure of ConA
After Alvarez-Manilla

11. Functions of Plant lectins
Little is known
In legume seeds can comprise up to 30% of the total protein
They are expressed in other parts of the plant
Nodulation factor in roots
After Alvarez-Manilla

12. Functions of Plant lectins (cont)
May function as defense against pathogens
Some lectins posses other activities besides carbohydrate binding
RCAII (Ricin) RNA-N-glycosidase
DBA has an adenine binding site in addition to CRD
After Alvarez-Manilla

13. Uses of Plant lectins Agglutination of cells and blood typing
Cell separation and analysis
Bacterial typing
Identification and selection of mutated cells with altered glycosylation
Toxic conjugates for tumor cell killing
Cytochemical characterization/staining of cells and tissues
After Alvarez-Manilla

14. Uses of Plant lectins (cont)
Mitogenesis of cells
Mapping neuronal pathways
Purification of glycoconjugates
Assays of glycosyltransferases and glycosidases
Defining glycosylation status of target glycoconjugates
After Alvarez-Manilla

15. Affinity/Avidity/Multivalency

16. Affinity determines approach

17. Washing can kill you

18. Characterizing lectin binding
Equilibrium dialysis against labeled hapten
Equilibrium binding, stop by PEG with centrifugation (solubilized receptor)
Equilibrium binding, stop by filtration (membranes)
Multivalent ligands
Multivalent receptor probes
Biacore realtime kinetics
Cell adhesion, flow under shear to immobilized glycan or receptor
Cell adhesion, static adhesion to immobilized glycan
X-ray co-crystallography, NMR, and MS mapping of relevant contacts and protein dynamics

19. Static cell adhesion, controlled force
Blackburn, C., 1982

20. Adhesive specificity determined by controlled detachment
Blackburn, C., 1985

21. Adhesive strength by controlled detachment
Blackburn, C., 1985

22. Binding specificity to resolved glycoconjugates
Tiemeyer, M., 1991

23. General Principles
Lectins generally bind with low affinity but achieve high avidity through multi-valency
A relatively small set of protein motifs have been identified as lectins
Lectin motifs comprise distributed sequence similarities and structural homologies; extended primary amino acid sequence conservation is not generally associated with lectin-like activities
Methods for lectin characterization must consider affinity, valency, and avidity
The development of multivalent probes, as well as methods for determining static and dynamic adhesion have been instrumental for defining lectin binding specificity and lectin function