Lectins

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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.
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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)
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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.
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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.
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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.
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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) Canaval
ia 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
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Lectin Protocol for EM
Osmium and Epon/Spurrs 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
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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
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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
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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
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Enzyme Labeling Reaction
Enzyme (recycled)
Exogenous substrate insoluble rxn
product Electron dense product (preci
pitant)
Trapping agent
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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)
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Enzymes as organelle markers
Enzyme General Location Acid
Phosphatase Golgi, ER, Lysosomes Alkaline
phosphatase Plasma membrane Catalase Peroxisom
e (microbody) Cytochrome oxidase Mitochondria T
hiamine pyrophosphatase Golgi
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Tissues fixed 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 for transmission electron microscopy.
Ultrathin sections post-stained with uranyl
acetate and lead citrate.
Yang, Berin, Yu, Conrad and Perdue, J Clin
Invest, 2000, 106879-886
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Thyamine pyrophosphate in Golgi
Peroxidase in multivesicular body
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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)
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Acid phosphatase in thick sections
200 kV
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Acid phosphatase in lysosomes
Acid phosphatase combined with immuno-localization
in lysosomes
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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.
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Tannic acid fix on human sperm
Actin filaments decorated with myosin
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In Situ hybridization as a localization technique
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In Situ hybridization labeling of HIV proteins