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Lectins and beyond

3/17/09

Lectins

–Lectins of plants–Lectins of animals–Lectins in signaling and immune response/inflammation

Lectins

• Carbohydrate binding proteins that are not antibodies orenzymes

• Bind with high specificity– Latin: lectus, meaning to gather or select– Relatively high dissociation constants (ca 100 µM)– Carbohydrate recognition domains are small

• Most lectins are multivalent

After Alvarez-Manilla

SolubleGlycan-Binding

Proteins

Glycan binding proteins “Translate” the Glycome

After Varki

Lectins are present in all organisms

• Virus----- Influenza• Bacteria ----- binding to hosts during pathogenesis• Plant

– Many have been purified and characterized– Physiological function is unknown

• Animal– Several proteins with a wide variety of functions

After Alvarez-Manilla

Plant Lectins

• Leguminosae– 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

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Structure of Plant lectins

• Compact β-barrel, no alpha helices– Anti-parallel beta-sheets

• Many require metals (leguminosae)– Ca and Mn– Metals do not participate directly in the binding

but are required for tertiary structure

After Alvarez-Manilla

Fig.29.1

Structure of concanavalin A (ConA), a legume seed lectin.

After Varki

Examples of N-glycans recognized by concanavalin A (ConA) from Canavalia ensiformis andGalanthus nivalis agglutinin (GNA).

After Varki

Examples of types of N-glycans recognized by L-PHA, E-PHA, and DSA.The determinants required for binding are indicated in the boxed areas.

After Varki

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

Functions of Plant lectins (cont)

• May function as defense against pathogens

• Some lectins possess other activities besides carbohydratebinding– RCAII (Ricin) RNA-N-glycosidase

– DBA has an adenine binding site in addition to CRD

After Alvarez-Manilla

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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

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

-shallow binding pockets-metal coordination

After Schnaar

Animal lectins are everywhere

After Schnaar, R.L. After Schnaar, R.L.

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After Varki

C-type Lectins

Mannose Binding Protein (C-type lectin)

• Mannose bindingprotein is trimericand each subunitbinds to mannose

• Notice thatbinding occurs inshallow pocketsnear the surface

After Esko

C-type Lectins

After Varki

After Varki After Varki

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After Varki After Esko

Galectins (S-type)

Comparison of the subunit structures of soybean agglutinin (left) complexed with a pentasaccharidecontaining Gal_1-4GlcNAc-R and human galectin-3 (right) complexed with lactose

Both lectins display a related _-barrel configuration.

After Cummings

After Cummings After Cummings

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Possible biosynthetic routes for galectins in animal cells.

After Cummings

Functional interactions of galectins with cell-surface glycoconjugates and extracellularglycoconjugates can lead to cell adhesion and signaling. Interactions with intracellularligands may also contribute to regulation of intracellular pathways.

After Cummings

A list of known and putative functions and biological activities of galectinstoward cells in the immune system.

After Cummings

I-type Lectins (Siglecs)

Domain structures of the known Siglecs in humans and mice.

After Varki

From: Crocker P, Paulson J. & Varki, A. Nature Reviews Immunol. 7:255-266, 2007.

Biological Interactions Involving Siglecs

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Biological functions mediated by sialoadhesin: Interactions of sialoadhesin onmacrophages with cells and pathogens.

(Right) Red staining shows ring of sialoadhesin expressed by macrophages in marginalzone of spleen and green staining shows Siglec-H on the plasmacytoid dendritic cells

After Varki

Biological functions mediated by myelin-associated glycoprotein (MAG)

After Varki

Sheikh, KA, et al. (1999) PNAS 96, 7532

MAG ligand loss leads to myelin degradation

Lectin binding characteristics

–Shallow binding/recognition sites–Distributed protein sequence motifs–Binding mediated by salt bridges, metal coordination, Van

der Waals interactions, aromatic stacking, hydrogenbonding

–Generally low affinity–Strong binding results from multivalency, BUT……

Cholera Toxin

• Cholera toxin exists asa AB5 complex, each Bsubunit binds to a singlemolecule of GM1

• The glycan binds toshallow pockets nearthe surface

• Kd of monomer for GM1~40 nM, very unusual(usually µM to mM)

After Esko

Cholera Toxin

• Multivalency raises affinityof interaction to ~40 pM

After Esko

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GlcGal

GalNAc

Gal

Neu5Ac

– Electrostatics

– H-bonding

– van der waals interactions

– Tyr, Phe, or alkyl side chains

– Water and divalent cations canact as bridging groups thatincrease selectivity

• Only 3 sugars arebound

• Selectivity is high,since binding occurswith great precision

After Esko

Affinity/Avidity/Multivalency

Affinity determines approach

WashMethod

Wash Time WashEfficiency

Ease Problems

Filtration 20 – 30 sec High Easy Filterbinding

Centrifugation 0 – 5 min Moderate Moderate PelletTrapping

Inert Phase 10 sec High Moderate Effect ofinert phase

EquilibriumDialysis

0 sec None Difficult LowSignal/Noise

Washing can kill you

pKD t0.5 t0.111 19 hr 2.9 hr10 1.9 hr 17 min9 11.5 min 1.7 min8 1.1 min 10 sec7 6.9 sec 1.0 sec6 0.7 sec 0.1 sec

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

Static cell adhesion, controlled force

Blackburn, C., 1982

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Adhesivespecificitydeterminedbycontrolleddetachment

Blackburn, C., 1985

Adhesivestrength bycontrolleddetachment

Blackburn, C., 1985

Screening for lectins, high throughput, no assumptions

After Varki

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 acidsequence conservation is not generally associated withlectin-like activities

–Methods for lectin characterization must consider affinity,valency, and avidity

–The development of multivalent probes, as well as methodsfor determining static and dynamic adhesion have beeninstrumental for defining lectin binding specificity andlectin function

To define the paradigm by which glycan bindingproteins modulate cell function, especially of immunecells and immune responses

The Prime Directive--Consortium for FunctionalGlycomics

General Principles

–Cell-surface glycans participate in all arms of theimmune system

–Glycan recognition is an integral component of innate(antibody-independent) immunity

–Specific glycan structures modify humeral immune(antibody-dependent) responses

–Differentiation of immune cell types requiresappropriate glycan expression

–Cell-cell interactions that lead to immune cellactivation are modulated by carbohydrate

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Selected topics

–Thymic selection of self vs. non-self–Innate immunity–Generation and function of the immune synapse–Carbohydrate-dependent antibody structure–Regulation of antibody production and B-cell

responsiveness by glycan

What does restricted mean? MHC I, MHC II, Cd1d?

Self vs. non-self selection Core-2 synthesis generates ligands for Galectins

Galectin binding induces apoptotic signals C2GlcNAcT activity in relation to thymic selection

–C2GlcNAcT expression by naïve T-cells in the thymiccortex increases with residence time and decreasesas the differentiating T-cell migrates to the medulla

–Strong TCR activation by self-bound MHC results inretention of the naïve T-cell in the cortex andmaintenance of C2GlcNAcT activity (negativeselection)

–Weak TCR activation results in migration of the T-cellto the thymic medulla and down-regulation ofC2GlcNAcT activity (positive selection)

–C2GlcNAcT activity generates substrates for Galectinbinding, leading to apoptosis of cortical T-cells

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Tissue surveillance leads to the activation of T-cells and the initiation of innate immune responses

Professional antigen presenting cells sit at the interfacebetween innate and adaptive responses

Innate immunity first then discuss antigen presentation

Innate immune responses detect the presence of pathogen, ornon-self molecular patterns--frequently these are glycans

Caroff, M., 2002 Caroff, M., 2002

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Caroff, M., 2002 Takeda, K., 2003

Activation of dendritic cells by various TLR ligands produces subsets of cytokines that activate adaptive responses

Iwasaki, A., 2004

Antigen presentation and the adaptive immune response

After Varki, A.

How is antigen presented?

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Glycans are essential for peptide loading onto MHC-I

Rudd, P., 2001

The immunological synapse is the communication point foractivation of specific T-cells

Dustin, M., 2000

Red/brown = cell adhesion molecules, integrins, Ig-CAMGreen/yellow = TCR, MHC I/IIWhite = outlines of T-cell on an APC

Dustin, M., 2000

The synapse must accommodate glycans

Rudd, P., 2001

Galectins may organize components of the synapse, providinga regulatory matrix that modulates signaling levels--GlcNAcTVKO has overactive signaling

Lowe, J., 2001

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Altered glycosylation of CD43 (leukosialin) modulates the synapse

Tsuboi, S., 2001

T-cell activation modulates B-cell responses

Th1--IFNγ, IL12, microbial responses,auto-immunity

Th2--IL4, 5, 6, 9, 10, 13, parasiteresponses, asthma, allergy

Three Possible Models for Functions of CD22-SialicAcid Interactions

Collins, B., 2002

Proposed Biological functions mediated by CD22: CD22 glycan-dependent homotypicinteractions in equilibrium with CD22–BCR interactions. IgG1 glycans are essential for antibody function

Rudd, P., 2001

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Under galactosylated IgG1 is recognized by MBP, leading to complement activation--mechanism of pathology in RA and SLE?

Rudd, P., 2001

B-cells and glycans

–Subsets of antibodies require appropriateglycosylation for function

–B-cell activation is modulated by carbohydrate-lectininteractions

General Principles related to glycansand immune function

–Cell-surface glycans participate in all arms of theimmune system, where they:

Mediate or regulate cell adhesionModulate cell signaling events

–By regulating signaling and adhesion, progenitor cellsare driven toward specific differentiation/activation byglycan expression

–Immunoglobulins, especially, require specific N-linkedglycans for stability and activity