Lectin blot overlay Lectin blot overlay

Methods and Experiments in BiochemistryMethods and Experiments in Biochemistry

Hungwen ChenHungwen Chen

R609, Institute of Biological Chemistry, Academia Sinica.R609, Institute of Biological Chemistry, Academia Sinica.hwchen@gate.sinica.edu.twhwchen@gate.sinica.edu.tw

Edited by Janway Chen

1. The avidin-biotin system1. The avidin-biotin system

2. Lectins2. Lectins

3. Wheat Germ Agglutinin Endo H and PNGase F 3. Wheat Germ Agglutinin Endo H and PNGase F

Lectin blot overlay Lectin blot overlay

Edited by Janway Chen

Biotin is a small molecular weight (244 dalton), water-soluble vitamin found in tissues and blood. Since biotin is a relatively small molecule, it can be conjugated to many macromolecules without altering their size, physical characteristics and biological activities. When a protein is biotinylated, it is advantageous to biotinylate it in a manner which will maintain its biological activity.

1. The Avidin-Biotin System1. The Avidin-Biotin System

●Biotin binds with high affinity to avidin, a 66 K dalton protein found in egg whites.

●The avidin-biotin interaction is the strongest known noncovalent biological recognition (Ka =1015 M-1 ) between protein and ligand.

●The bond formation between biotin and avidin is very rapid and once formed is unaffected by extremes of pH, organic solvents and other denaturing agents. The strength of the avidin-biotin interaction has provided researchers with a unique tool for use in immunoassays, receptor studies, immunocytochemical staining and protein isolation. Labeled streptavidin may be used to detect biotinylated lectins and antibodies which in turn would bind to the appropriate glycoprotein or antigen.

● Biotinylated lectins provide ideal intermediates for examining glycoconjugates using the avidin-biotin system.

● A particular glycoconjugate may be detected by addition of the appropriate biotinylated lectin, followed by avidin conjugated with fluorochromes, enzymes, or electron microscope (EM) markers in ELLA (Enzyme-Linked Lectin Assay), Western blotting and immunohistochemistry studies.

● ELLA is a procedure similar to ELISA (Enzyme-Linked Immunosorbent Assay) in which an enzyme-lectin complex is utilized for the detection and quantitation of glycoproteins.

2. Lectins2. LectinsThe sugar binding specificities of lectins have made them fundamental tools in glycoprotein research since sugars present in glycoconjugates can be analyzed selectively.

● In glycoprotein studies, it is more advantageous to use streptavidin than avidin since avidin is a glycoprotein. Streptavidin does not contain any carbohydrate, which eliminates any doubt about extraneous carbohydrate.

● There are three major advantages of using streptavidin-biotin interactions in both blotting and ELLA procedures:

a. They increase the sensitivities of these assays. b. The same streptavidin and biotin reagents may be used to an

alyze different glycoconjugates. c. The biotinylation of a lectin does not appear to alter its bindin

g properties.

● When bound to immobilized streptavidin, biotinylated lectins may be used to purify glycoproteins.

● Lectins can selectively agglutinate specific blood groups and are used for isolation and characterization of blood group-active saccharides. Lectins are used to discern the location and structure of blood group-active molecules in the erythrocyte membrane. The agglutinated cells can be isolated using a biotinylated lectin. The complexes are bound to immobilized streptavidin. The lectin-bound cells are completely eluted with high concentrations of simple sugars.

● Lectins are useful in affinity chromatography applications. Immobilized lectins are useful for preparation and purification protocols. When using immobilized lectins, a mixture of glycoproteins and polysaccharides is obtained. The resulting preparation, although not pure, will be enriched in glycoproteins. This may be useful in the isolation of minor glycoprotein components or as a step in the purification of membrane receptors. Different immobilized lectins can be used in a prescribed order to obtain samples of higher purity.

● Based on the binding pattern of a particular glycoprotein to a series of lectins, it is possible to suggest a nominal structure for the protein.

● Many lectins are stable in detergents, making them useful in the purification of membrane proteins. When purifying membrane glycoproteins, the detergent used for solubilization must be considered. Nonionic detergents, such as Nonidet P-40 and Triton X-100, do not significantly affect the ability of immobilized lectins to bind glycoproteins. Therefore, they do not interfere in the affinity chromatography of the sample. However, an anionic detergent such as SDS may denature or dissociate the lectin, leaving it inactive.

Edited by Janway Chen

● In some cases, direct conjugation of a lectin to a support may cause steric hindrance or may alter the binding site and prevent the lectin from binding carbohydrates. In these situations, the lectin may be biotinylated and bound to immobilized streptavidin. Lectins have been categorized based on carbohydrate specificity:

a. Mannose/glucose

b. N-Acetylglucosamine

c. N-Acetylgalactosamine/galactose

d. L-Fucose

e. Sialic Acid

f. “Complex” carbohydrates

Edited by Janway Chen

● The lectin is isolated as a mixture of 3 or 4 molecular species: Isolectins I, IIa, IIb and III that differ in electrophoretic mobility. The lectin activity is distributed among 4 isolectins: 35% of I, 50% of IIa, 5% of IIb and 10% of III.

● WGA fractions I, IIa and III are indistinguishable upon isoelectric focusing giving a sharp band at pH 8.7 ± 0.3. IIb gives a sharp band at pH 7.7 ± 0.3. The amino acid composition of the four forms are nearly identical except that fraction 1 contained no histidine whereas the other three forms each contained two histidine residues per subunit. All the forms are rich in glycine (25%) and every fifth amino acid residue is a half cysteine, but no sulfhydryl residues are present.

3. Wheat Germ Agglutinin3. Wheat Germ Agglutinin

Wheat Germ (Triticum vulgare) Agglutinin (WGA) has been purified both by conventional and various affinity chromatography methods. The lectin is a 36,000 molecular weight protein, carbohydrate-free, dimeric protein composed of two identical subunits. Subunit molecular weights range from 17,000 to 24,000 daltons. The amino acid sequence provides an estimate of 21,600 daltons, based on 171 amino acids per polypeptide chain.

● The carbohydrate-binding specificity has been determined by a variety of techniques. These studies show that of the simple sugars tested only N-acetylglucosamine binds to WGA. N-Acetylmannosamine does not bind and N-acetylgalactosamine interacts very weakly with the lectin. Wheat Germ Agglutinin exhibits little or no anomeric preference for either the methyl a- or b-glycoside of N-acetylglucosamine. Glucosamine itself is not inhibitory; apparently a charged amino group is incompatible with WGA binding. Substitution of a methyl group at the O-4 or O-6 position of N-acetyl-glucosamine had no effect on inhibitory activity, whereas similar substitution at the O-3 position gave an inactive derivative.

● Specific precipitation of WGA by glycoproteins, polysaccharides, glycolipids, bacterial cell wall, peptidoglycan and synthetic model substrates have been reported in literature. Sialidase treated animal cells have been shown to reduce or abolish binding of WGA suggesting that this lectin could bind to sialyl groups of glycoproteins.

● The specific binding of N-acetylneuraminic acid to WGA is based on the similarity in configuration of this sugar to N-acetylglucosamine at positions C-2 (N-acetamido group) and C-3 (hydroxyl group) of the pyranose ring. These are the positions critical to productive contact with the WGA combining site. Although N-acetylgalactosamine also possesses the above configurational determinants it binds to WGA only weakly because it contains an axial C-4 hydroxyl group.

Edited by Janway Chen

● Successful application of oligosaccharide-cleaving enzymes in the cell biology of glycoproteins may focus on the structure-function relationships of the covalently attached carbohydrate present on isolated, purified glycoproteins or may follow the biosynthesis and modification of glycoprotein glycans as probes of cellular processes, including targeting, transport, and turnover.

4. Enzymatic approaches for studying glycoproteins4. Enzymatic approaches for studying glycoproteins

Edited by Janway Chen

(1) GlcNAc-Asn sugar chains (-Asn-X-Ser/Thr-, X represents any amino acid except perhaps Pro or Asp)

High Mannose

Hybrid

ComplexBiantennary

Biantennary

Triantennary

(2) Endoglycosidases and Glycoprotein-Asn Amidases

(3). Application

Edited by Janway Chen