ESSENTIALS OF GLYCOBIOLOGY LECTURE 24 MAY 9, 2002 Richard D. Cummings, Ph.D. University of Oklahoma...
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Transcript of ESSENTIALS OF GLYCOBIOLOGY LECTURE 24 MAY 9, 2002 Richard D. Cummings, Ph.D. University of Oklahoma...
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ESSENTIALS OF GLYCOBIOLOGY
LECTURE 24
MAY 9, 2002
Richard D. Cummings, Ph.D.University of Oklahoma Health Sciences Center
College of MedicineOklahoma Center for Medical Glycobiology
“THE PLANT LECTINS”
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Definition of a Lectin -
“A protein (other than an anti-carbohydrate antibody) that specifically recognizes and binds to glycans without catalyzing a modification of the glycan.”
The first lectins identified were derived from plants, specifically leguminous seeds.
Until recently, it was thought that a lectin must be multivalent and soluble.
But some monovalent, monomeric lectins, and many membrane-bound lectins, are now known.
“THE PLANT LECTINS”
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1888 H. Stillmark Ricinus communis plant extracthas hemagglutinating properties
1890 P. Ehrlich Lectins used as antigens in earlyImmunological studies
1908 K. Lansteiner & Different hemagglutinating H. Raubitsheck properties in various plant seeds
1919 J. Sumner Crystallization of Con A
1936 J. Sumner Lectins bind sugar - Con Aprecipitates glycogen
Date Investigators DiscoveryHistory of Plant Lectins
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Lycopersicum esculentum(tomato)
Lens culinaris(lentil)
Ricinus communis(castor bean)
Datura stramonium(jimsonweed)
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1940 W. Boyd, Lectins specific for some human R. Reguera & blood group antigens
K.O. Renkonen
1952 W. Watkins & Use of lectins and glycosidases to W. Morgan prove that blood group antigens
are sugars and to deduce thestructures of the antigens
1954 W. Boyd & The name lectin is proposed to E. Shyleigh replace hemagglutinin
Date Investigators Discovery
History of Plant Lectins
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1960 P.C. Nowell Red kidney bean lectin P. & J.C. Aub vulgaris mitogenic for resting
lymphocytes
1960’s M. Burger Lectins preferentially1970’s G. Nicolson agglutinate some animal tumor
cells
1980’s Kornfeld(s) Use of immobilized lectins Osawa to analyze animal Kobata glycoconjugates Cummings
1980’s D. Kabelitz Discovery that plant lectins1990’s D.J. Gee induce apoptosis
K. Schweizer
Date Investigators Discovery
History of Plant Lectins
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Lectin group Structure of CRD Length
Calnexin Unknown ? L-type -sandwich ~230
(Legume lectin-like) P-type Unique -rich structure ~130
(Phosphomannose) M-type Unique -helical ~500
(mannosidase-related C-type Unique mixed /ß structure ~115
(Ca2+-dependent) Galectins -sandwich ~125 I-type Immunoglobulin superfamily ~120 R-type -trefoil (plants and animals) ~125
(Ricin related)
SOME FAMILIES OF LECTINS DISTINGUISHED BY 3º STRUCTURE
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• 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• Mitogenesis of cells• Mapping neuronal pathways• Purification and characterization of glycoconjugates• Assays of glycosyltransferases and glycosidases• Defining glycosylation status of target glycoconjugates
Uses of Plant Lectins
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METAL BINDING SITES- - -V- - -D- -
LIV
STAG
EQV
FLI
ST
-Q-V-V-A-V-E-F-D-T-F-R-N- SBA-L-T-V-A-V-E-F-D-T-C-H-N- Lima bean lectin
-V-L-D-D-W-V-S-V-G-F-S-A- Lima bean lectin-S-L-P-E-W-V-R-I-G-F-S-A- SBA
Conserved Motif In C-terminal Domain
N-TERMINI
A-E-T-V-S-F-S-W-N-K-F-V-P-K-Q-
A-E-L-F-F-N-F-Q-T-F-N-A-A-N-Lima bean lectin Phaseolus limensis
SBA - Soybean agglutinin (Glycine max)
Primary Structural Motifs in Leguminous (L-type) Plant Lectins
Red = invariant residues
- -x- - -V-x- -G- - - LIV
EDQ
FYWKR
LIV
FL
ST
1
1
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Legumes
Grains
Diverse
Primarily Amino Sugars
(GlcNAc/NeuAc)
25-30
~18
2 or 4
2
1
2
Class MonosaccharideSpecificity
Subunit MW(kDa)
SubunitsBindingSites perSubunit
Classifications of Some Plant Lectins
Legumes
Grains
Variable
Variable
No
Yes
Mn2+, Ca2+
No
Class Glycosylation -S-S-Bonds
Metals
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Bovine Galectin-1 DimerCon A Dimer
Similarities in Protein Folding Between Galectins and Legume L-type Lectins
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Crystal structure of artocarpin lectin from the jack fruit (Artocarpus integrifolia) (left - monomer; right - tetramer)
Protein Folding in L-type Lectins
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Structure of L-type Tetrameric ConA at 2.35 Å.
The trimannoside ligand is indicated in space-filling mode and the coordinated Ca2+ and Mn2+ are shown as the large green balls and small pink balls, respectively. The crystal structure was originally reported as a complex of ConA and a trimannoside ligand by Naismith and Field (Naismith J.H. and Field R.A. 1996. Structural basis of trimannoside recognition by concanavalin A. J. Biol. Chem. 271: 972–976).
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Ribbon representation showing the overall structure of Dioclea guianensis Seed Lectin tetramer and the relative location of the metal ions in the four subunits. The Mn2+ (green) and Ca2+ (yellow) of the canonical (S1 and S2) metal-binding site are shown as spheres. The secondary sub-sites for the Ca2+ /Cd2+ (S3) and Mn2+ (S5) are depicted as purple and blue spheres, respectively. (Ref: Wah et al, (2001) J. Mol. Biol. Vol. 310
Çrystal Structure of the L-type Dioclea guianensis Seed Lectin
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Crystal Structure of “Grain-type” Wheat Germ Agglutinin (Isolectin 2) Dimer in Complex With N-Acetylneuraminyllactose
Wright CS (1990) 2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose--wheat germ agglutinin isolectin complexes J Mol Biol 215, 635-651
sialyllactoseSugar binding site
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Because of their multivalency and oligomeric structures, many plant lectin can cross-linking can
precipitate glycoproteins and agglutinate cells
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R R
R
R R
R
R R
RDrosophila Lectins
Bacterial Lectins
Bacterial Hydrolases
Ricin/Plant Toxins
GalNAc Transferases
Mannose Receptor Family
R CCCCCCCC
R R-type CRD
R-type CRD
GalNAcT DomainHydrolase Domain C C-type CRD
TM domainFibronectin domain
Ricin-typeR-type Lectins
- -trefoil proteins
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Comparisons between Cys-MR (R-type domain in the mannose receptor) and other -trefoil proteins - Cys-MR, a portion of the ricin B chain (residues 1–136 with N-linked carbohydrates omitted; and human aFGF (from Liu Y et al. (2000) J. Exp. Med., 191:1105-16)
Structures of R-type Lectins
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The plant toxin ricin consists of two disulfide-linked polypeptides with different functions. The A-chain enters the cytosol and inactivates the ribosomes enzymatically (the A chain of ricin has RNA N-glycosidase activity to cleave a specific adenine base from ribosomal RNA), whereas the B-chain has lectin properties and binds to carbohydrates at the cell surface. (The structures have been obtained from the PDB protein data bank (ricin: 1DMO; Shiga toxin:2AA1), and are based on work published by Rutenber et al. (1991) and Fraser et al. (1994).)
Crystallographic structures of ricin (A) and Shiga toxin (B)
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This binding is a requirement for translocation of the A-chain to the cytosol. The bound toxin is endocytosed and transported retrograde through the Golgi apparatus to the endoplasmic reticulum where it appears to be translocated to the cytosol by the sec61p complex. (ref: Olsnes S, Kozlov JV. (2001) Ricin. Toxicon 39(11):1723-8). The cytosolic target of ricin and Shiga toxin is the 28S RNA of the 60S ribosomal subunit (Endo et al., 1987). Reduction of the disulfide bond connecting the A- and B-moieties of ricin is required for optimal enzymatic activity.
Crystallographic structures of ricin (A) and Shiga toxin (B)
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During biosynthesis, some of the leguminous lectins are proteolytically cleaved to generate a b-chain, corresponding to the amino terminus, and an a-chain, corresponding to the carboxyl terminus.
For example, jacalin lectin, from the jackfruit Artocarpus heterophyllus, is a tetrameric two-chain lectin (65 kD) (molecular mass 65 kD) with an a-chain of 133 amino acid residues and a b-chain of 20-21 amino acid residues.
An exceptional situation occurs with the well-known lectin Con A from jack beans (Canavalia ensiformis).
Con A is generated as a glycoprotein precursor, but it is proteolytically processed; the propeptide with the N-glycan is removed; the two chains are transposed and rejoined with the formation of a new peptide bond to generate the intact protein.
Thus, with regard to other lectins, the mature amino terminus of ConA corresponds to an a-chain and the carboxyl terminus corresponds to a b-chain.
In sequence alignments with other lectins, ConA exhibits what is called “circular” homology.
Lectin Biosynthesis
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Seed storage proteins Aid in maintaining seed dormancy Defense against fungal, viral, and bacterial pathogens Defense against animal predators Symbiosis in lugumes Transport of carbohydrates Mitogenic stimulation of embryonic plant cells Elongation of cell walls Recognition of pollen
Biological Functions of Plant Lectins
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The roots of the legume Dolichos biflorus contain a lectin/nucleotide phosphohydrolase (Db-LNP) that binds to the Nod factor signals produced by Nod genes in rhizobia that nodulate this plant.
Db-LNP is differentially distributed along the surface of the root axis in a pattern that correlates with the zone of nodulation of the root. Db-LNP is present on the surface of young and emerging root hairs and redistributes to the tips of the root hairs in response to treatment of the roots with a rhizobial symbiont or with a carbohydrate ligand. (Ref: Kalsi G, Etzler ME. (2000).
Localization of a lipo-chitin oligosaccharides (LCOs), or Nod factors and Nod factor-binding protein in legume roots and factors influencing its distribution and expression. Plant Physiol 124(3):1039-48).
Nod C encodes a GlcNAcT to synthesizes the chitin glycan; Nod B catalyzes the de-N-acetylation; Nod A catalyzes N-fatty acylation
Plant Lectin Function in Nitrogen Fixation/Rhizobial Infection
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NHFatty Acid
OH
ROHO
NAc
OH
OH
HO
H3C
OH
NHFatty Acid
OH
HO
NAc
OH NHHFatty Acid
HO
OH
HO
H3C
OR
OHHO
A
B
CD
EG
F
Structure of lipo-chitin oligosaccharides in the pooled HPLC fractions 7 and 8 of Mesorhizobium loti strain NZP2213. Monosaccharide residues are labeled A-G. R1, predominantly C20:1 and C18:0, with other minor fatty acids; R2, carbamoyl NH2CO-; R3, acetyl or H. Olsthoorn et al, (1998) Biochemistry 37(25):9024-32
Plant Lectin Function in Nitrogen Fixation/Rhizobial Infection
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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 Mitogenesis of cells Mapping neuronal pathways Purification and characterization of glycoconjugates Assays of glycosyltransferases and glycosidases Defining glycosylation status of target glycoconjugates
Some Uses of Plant Lectins
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Agarose bound* Aleuria Aurantia Lectin (AAL)
Alkaline Phosphatase conjugated Aleuria Aurantia Lectin (AAL)
Biotinylated Aleuria Aurantia Lectin (AAL)
Unconjugated Aleuria Aurantia Lectin (AAL)
VECTREX AAL
VECTREX AAL Binding and Elution Kit
Example of a Catalog Listing (Vector Labs) Lectin Products
Example - Aleuria Aurantia Lectin (AAL)
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Con A
LCA LCA
L-PHA L-PHA
Fraction Number
SNA
Further Purification on Other Lectins, HPLC, etc.
SNA
Quantity of Glycan
Serial Lectin Affinity Chromatography for Fractionation and Purification of Complex Carbohydrates
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[Hapten: 0.1 M -Methyl-Glc]
[Hapten: 0.1 M -Methyl-Man]
[Hapten: 0.5 M -Methyl-Man]
Lectin Recognition of Glycans
Mannose-Binding in N-Glycans
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Phaseolus vulgaris
leukoagglutinin (L4-PHA)Hapten: 0.4 M GalNAc
Datura stramonium agglutinin (DSA) (weakly)
Hapten: 10 mg/mlChitotriose
Phaseolus vulgaris
erythroagglutinin (E4-PHA)
Hapten: 0.4 M GalNAc
Man-GlcNAc-GlcNAc-AsnGal GlcNAc Man
1,4Gal GlcNAc
Man-GlcNAc-GlcNAc-Asn
1,6
Man-GlcNAc-GlcNAc-Asn
GlcNAc
1,4
Bound By
1,4 1,2
Gal GlcNAc Man1,4 1,2
1,4
Gal GlcNAc1,4
Gal GlcNAc Man1,2
Gal GlcNAc Man1,4 1,2
Gal GlcNAc Man1,4 1,2
Gal GlcNAc Man1,4 1,2
Lectin Recognition of Glycans
1,4
Galactose-Binding in Complex-type N-glycans
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Hapten for both: 0.1 M lactose
Hapten: 10 mM raffinose
Hapten: 50 mM GalNAc
Erythrina cristagalli lectin (specific for Gal4GlcNAc-R)
Ricinus communis agglutinin (RCA-I) (binds better to Gal4GlcNAc-R than
To Gal3GlcNAc-R )
Lectin Recognition of Glycans Galactose-Binding in Complex-type N- and O-glycans, and Glycosphingolipids
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Hapten: 0.2 M Fuc
Hapten: 0.2 M Fuc
Hapten: 0.2 M -methyl-Man
Hapten: 10 mM Fucose
Lectin Recognition of Glycans
Fucose-Binding in Complex-type N- and O-glycans, and Glycosphingolipids
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[Hapten:10 mg/ml Chitotriose]
[Hapten: 0.1 M GlcNAc]
Lectin Recognition of Glycans
N-Acetylglucosamine-Binding in Complex-type N- and O-glycans, and Glycosphingolipids
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[Hapten: 50 mM Lactose]
[Hapten: 50 mM Lactose]
Lectin Recognition of Glycans
Sialic acid-Binding in Complex-type N- and O-glycans, and Glycosphingolipids
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[Hapten for all: 0.1 M GalNAc
[Hapten for all: 50 mM -Methyl-GalNAc]
Lectin Recognition of Glycans
Galactose- and N-acetylgalactosamine-Binding In O-glycans
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Con A
LCA LCA
L-PHA L-PHA
Fraction Number
SNA
Further Purification on Other Lectins, HPLC, etc.
SNA
Quantity of Glycan
Serial Lectin Affinity Chromatography for Fractionation and Purification of Complex Carbohydrates
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Add Alk.Phos.- Streptavidin-
Alk.Phos.-Streptavidin- Biotin-
Biotin-
Biotin-
Use of a lectin to assay a sialyltransferase
CMP
Gal1-4GlcNAc-R-
CMP-NeuAc
NeuAc2-3Gal1-4GlcNAc-R-
NeuAc2-3Gal1-4GlcNAc-R-
NeuAc2-3Gal1-4GlcNAc-R-
Add Biotinylated-MAL
Step 1
Step 2
Step 3
2-3-sialyltransferase
in an ELISA-type Method
COLOR