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Lectins

Lectins The term “lectin” is a general term that encompasses several families of proteins of non-immune origin that bind glycoconjugates that may or may not have a known function.

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Lectins

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  1. Lectins The term “lectin” is a general term that encompasses several families of proteins of non-immune origin that bind glycoconjugates that may or may not have a known function. Also known as agglutinins since original discoveries used agglutination of red blood cells (recognition of surface sugars) as a criteria. Many have secondary or even tertiary affinities to other sugars - rigorous controls required.

  2. 1. A lectin molecule contains at least two sugar-binding sites; sugar-binding proteins with a single site will not agglutinate or precipitate structures that contain sugar residues, so are not classified as lectins. 2. The specificity of a lectin is usually defined by the monosaccharides or oligosaccharides that are best at inhibiting the agglutination or precipitation the lectin causes. 3. Lectins occur in many types of organisms; they may be soluble or membrane-bound; they may be glycoproteins. 4. Sugar-specific enzymes, transport proteins and toxins may qualify as lectins if they have, multiple-sugar binding sites. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), and Nomenclature Commission of IUB (NC-IUB)

  3. General Classes of Lectins Animal 1) Galectins share galactose-specificity. 2) Ca-dependent (C-type) animal lectins form an extremely large family, composed of members having diverse structures and functions. Selectins are a subfamily that have a specific function in leukocyte adhesion to endothelial cells through sialyl-LewisX recognition. Collectins have a unique structure consisting of a C-type lectin domain and a collagen-like domain. They are supposed to be involved in innate immunity.

  4. 3) Invertebrates (such as snails - e.g. Helix pomatia) are known to contain various lectins in their body fluids, possibly as body-protection factors. E.g. lectins from an echinoderm were found to show hemolytic activity. 4) Annexins have affinity to lipids with some having binding activity to glycosaminoglycans.

  5. Plant 7) The legume lectin family consists of a large number of members, such as Concanavalin A, with variable saccharide specificities comparable to C-type lectins. 8) Ricin was the first lectin investigated in Russia more than 100 years ago and is very toxic. It is now evident that ricin has many other homologous members which differ in either toxicity or sugar-binding specificities.

  6. Examples of commonly used Lectins Ricinis communis (RcAI and RcAII) - Castor bean a-D galactose, lactose (TOXIC!) Triticum vulgaris (WGA) - Wheat germ N-acetylglucosamine (trimers) (chitin) Helix pomatia (HpA) - Roman snail N-acetyl galactosamine (cellulose) Canavalia ensiformis (Concanavalin A) - Jack bean a-D mannose, a-D glucose Aplysia depilans (AGL) - Aplysia gonad lectin Galacturonic acids Ulex europaeus (UEA I) - Furze gorse seeds a -linked fucose

  7. Lectin Protocol for EM Osmium and Epon/Spurr’s resin can be used. 1. Sections collected on gold or nickel grids 2. (Optional) Block and rinse thoroughly 3. Expose lectin/gold conjugate to sections 4. Rinse thoroughly 5. Post-stain

  8. Lectin Controls Adsorption with primary affinity substrate/sugar Adsorption with secondary and/or tertiary affinity substrate/sugar Incubation with unlabeled lectin Use of non-specific label (e.g. BSA/gold complex) Incubation with stabilized colloidal gold suspension

  9. Lectin labeling of a germinating fungal spore Same regions (e.g. A) labeled with different lectins to show distribution of wall components top - chitin middle - cellulose bottom - N-acetyl galactosamine

  10. Cytochemical Labeling Identify carbohydrates, enzymes, proteins, etc… Intensity of catalytic activity can be determined. Chemical reactions require gold conjugate or electron opaque precipitate for visualization. Can be pre-embedding or post-embedding. DAB reaction showing peroxidase in lysosomes

  11. Enzyme Labeling Reaction Enzyme (recycled) Exogenous substrate insoluble rxn product Electron dense product (precipitant) Trapping agent

  12. Exogenous substrate - reacts with endogenous enzyme in tissue. The substance acted upon by the enzyme is the substrate. (e.g. H2O2 with peroxidase) Insoluble reaction product - deposition and retention of the product necessary for visualization. Trapping agent - insoluble heavy metal salt (e.g. lead); or the enzyme itself (e.g. HRP). Used to bind to and contain or trap the reaction product and make it directly visible. (e.g. DAB)

  13. Enzymes as organelle markers Enzyme General Location Acid Phosphatase Golgi, ER, Lysosomes Alkaline phosphatase Plasma membrane Catalase Peroxisome (microbody) Cytochrome oxidase Mitochondria Thiamine pyrophosphatase Golgi

  14. Tissuesfixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer(pH 7.4) Washed Incubated in 3,3'-diaminobenzadine tetrahydrochlorine(DAB)and H2O2 Further processed fortransmission electron microscopy. Ultrathin sections post-stained withuranyl acetate and lead citrate. Yang, Berin, Yu, Conrad and Perdue, J Clin Invest, 2000, 106:879-886

  15. Thyamine pyrophosphate in Golgi Peroxidase in multivesicular body

  16. Requirements for Enzyme Reactions *Preservation of tissue structure and enzymatic activity (formaldehyde and low glutaraldyhyde) *Maximization of reaction conditions (temp, pH, optimal substrate and trapping agent concentrations) *Facilitation of substrate penetration (typically limited to 100 mm - need to microtome prior to embedding) *Appropriate controls (lack of substrate and/or trapping agent) *Visualization of reaction product (view sections prior to any staining)

  17. Acid phosphatase in thick sections 200 kV

  18. Acid phosphatase in lysosomes Acid phosphatase combined with immuno-localization in lysosomes

  19. Miscellaneous Labeling Techniques Modified Periodic Acid Schiff Reaction Aldehyde groups are produced when periodic acid is used to oxidize carbohydrates. These are then reacted with alkaline silver solutions in which silver is deposited at the reaction site Alkaline bismuth stains certain polysaccharides incorporating the PA-Schiff reaction.

  20. Tannic acid fix on human sperm Actin filaments decorated with myosin

  21. In Situ hybridization as a localization technique

  22. In Situ hybridization labeling of HIV proteins

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