Transcript Lecture_24.pps

ESSENTIALS OF GLYCOBIOLOGY
LECTURE 24
MAY 9, 2002
Richard D. Cummings, Ph.D.
University of Oklahoma Health Sciences Center
College of Medicine
Oklahoma Center for Medical Glycobiology
“THE PLANT LECTINS”
“THE PLANT LECTINS”
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.
History of Plant Lectins
Date Investigators
Discovery
1888 H. Stillmark
Ricinus communis plant extract
has hemagglutinating properties
1890 P. Ehrlich
Lectins used as antigens in early
Immunological studies
1908 K. Lansteiner &
H. Raubitsheck
Different hemagglutinating
properties in various plant seeds
1919 J. Sumner
Crystallization of Con A
1936 J. Sumner
Lectins bind sugar - Con A
precipitates glycogen
Ricinus communis
(castor bean)
Lens culinaris
(lentil)
Datura stramonium
(jimsonweed)
Lycopersicum esculentum
(tomato)
History of Plant Lectins
Date Investigators
Discovery
1940 W. Boyd,
R. Reguera &
K.O. Renkonen
Lectins specific for some human
blood group antigens
1952 W. Watkins &
W. Morgan
Use of lectins and glycosidases to
prove that blood group antigens
are sugars and to deduce the
structures of the antigens
1954 W. Boyd &
E. Shyleigh
The name lectin is proposed to
replace hemagglutinin
History of Plant Lectins
Date Investigators
Discovery
1960 P.C. Nowell
& J.C. Aub
Red kidney bean lectin P.
vulgaris mitogenic for resting
lymphocytes
1960’s M. Burger
1970’s G. Nicolson
Lectins preferentially
agglutinate some animal tumor
cells
1980’s Kornfeld(s)
Osawa
Kobata
Cummings
Use of immobilized lectins
to analyze animal
glycoconjugates
1980’s D. Kabelitz
1990’s D.J. Gee
K. Schweizer
Discovery that plant lectins
induce apoptosis
SOME FAMILIES OF LECTINS
DISTINGUISHED BY 3º STRUCTURE
Lectin group
Structure of CRD
 Calnexin
Unknown
 L-type
b-sandwich
(Legume lectin-like)
 P-type
Unique b-rich structure
(Phosphomannose)
 M-type
Unique a-helical
(mannosidase-related
 C-type
Unique mixed /ß structure
(Ca2+-dependent)
 Galectins
b-sandwich
 I-type
Immunoglobulin superfamily
 R-type
b-trefoil (plants and animals)
(Ricin related)
Length
?
~230
~130
~500
~115
~125
~120
~125
Uses of Plant Lectins
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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
Primary Structural Motifs in Leguminous (L-type) Plant Lectins
N-TERMINI
1
A-E-L-F-F-N-F-Q-T-F-N-A-A-N-Lima bean lectin Phaseolus limensis
1
A-E-T-V-S-F-S-W-N-K-F-V-P-K-Q-SBA - Soybean agglutinin
(Glycine max)
Red =
invariant
residues
F
E Y
L
L
F S
- I -x- D - W -V-x- I -G- - - Conserved Motif In
L T C-terminal Domain
Q K
V
V
R
-V-L-D-D-W-V-S-V-G-F-S-A-S-L-P-E-W-V-R-I-G-F-S-A-
Lima bean lectin
SBA
S
E F
L
METAL BINDING
T
- I - -V- Q - L -D- S SITES
A
T
V I
V
G
-L-T-V-A-V-E-F-D-T-C-H-N- Lima bean lectin
-Q-V-V-A-V-E-F-D-T-F-R-N- SBA
Classifications of Some Plant Lectins
Class
Legumes
Grains
Class
Subunits
Binding
Sites per
Subunit
25-30
2 or 4
1
Primarily Amino
Sugars
(GlcNAc/NeuAc)
~18
2
2
Glycosylation
-S-SBonds
Metals
Monosaccharide Subunit MW
Specificity
(kDa)
Diverse
Legumes
Variable
No
Mn2+,
Ca2+
Grains
Variable
Yes
No
Similarities in Protein Folding Between
Galectins and Legume L-type Lectins
Con A Dimer
Bovine Galectin-1 Dimer
Protein Folding in L-type Lectins
Crystal structure of artocarpin lectin from the jack fruit
(Artocarpus integrifolia) (left - monomer; right - tetramer)
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).
Çrystal Structure of the L-type
Dioclea guianensis Seed Lectin
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
Crystal Structure of “Grain-type” Wheat Germ Agglutinin
(Isolectin 2) Dimer in Complex With N-Acetylneuraminyllactose
Sugar binding site
sialyllactose
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
Because of their multivalency and oligomeric
structures, many plant lectin can cross-linking can
precipitate glycoproteins and agglutinate cells
Drosophila Lectins
R
Bacterial Lectins
R
Ricin-type
R-type Lectins
- b-trefoil
proteins
R
R
Bacterial Hydrolases
R
Ricin/Plant Toxins
R
GalNAc
Transferases
R
R
R
R
C
C
C
C
C
C
C
C
Mannose Receptor Family
R-type CRD
R
R-type CRD
Hydrolase Domain
GalNAcT Domain
TM domain
C
C-type CRD
Fibronectin
domain
Structures of R-type Lectins
Comparisons between Cys-MR (R-type domain in the
mannose receptor) and other b-trefoil proteins - CysMR, 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., 191:110516)
Crystallographic structures of ricin (A) and Shiga toxin (B)
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 Nglycosidase 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)
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.
Lectin Biosynthesis
 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 bchain 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.
Biological Functions of Plant Lectins
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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
Plant Lectin Function in Nitrogen
Fixation/Rhizobial Infection
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. DbLNP 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):103948).
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
OH
OR
HO
HO
OH
H3C
OH
B
H3C
OH
HO
RO
OH
NAc
HO
NHFatty Acid
NAc
HO
C
A
OH
F
D
E
HO
NHFatty Acid
OH
OH
G
NHHFatty Acid
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
Some Uses of Plant Lectins
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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
Example of a Catalog Listing (Vector Labs)
Lectin Products
Example - Aleuria Aurantia Lectin (AAL)
 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
Serial Lectin Affinity Chromatography for Fractionation
and Purification of Complex Carbohydrates
Con A
Quantity of
Glycan
Fraction Number
LCA
L-PHA
L-PHA
LCA
SNA
SNA
Further Purification on Other Lectins, HPLC, etc.
Lectin Recognition of Glycans
Mannose-Binding in N-Glycans
[Hapten: 0.5 M a-Methyl-Man]
[Hapten: 0.1 M a-Methyl-Man]
[Hapten: 0.1 M a-Methyl-Glc]
Lectin Recognition of Glycans
Galactose-Binding in Complex-type N-glycans
Gal
Gal
b1,4
b 1,4
Gal
GlcNAc
GlcNAc
b1,4
b1,2
b 1,2
GlcNAc
Man
Man
Man-GlcNAc-GlcNAc-Asn
b 1,4
Bound By
Datura stramonium
agglutinin (DSA) (weakly)
Hapten: 10 mg/ml
Chitotriose
Gal b 1,4GlcNAc
b1,6
Gal b1,4 GlcNAc b1,2 Man
b 1,2
Gal b1,4GlcNAc
Man
Phaseolus vulgaris
Man-GlcNAc-GlcNAc-Asn
GlcNAc
b 1,2
Gal
GlcNAc
Man
b 1,4
Man-GlcNAc-GlcNAc-Asn
b1,4
b1,2
Gal
GlcNAc
Man
b 1,4
leukoagglutinin (L4-PHA)
Hapten: 0.4 M GalNAc
Phaseolus vulgaris
erythroagglutinin (E4-PHA)
Hapten: 0.4 M GalNAc
Lectin Recognition of Glycans
Galactose-Binding in Complex-type N- and
O-glycans, and Glycosphingolipids
Erythrina cristagalli lectin
(specific for Galb4GlcNAc-R)
Ricinus communis agglutinin (RCA-I)
(binds better to Galb4GlcNAc-R than
To Galb3GlcNAc-R )
Hapten for both: 0.1 M lactose
Hapten: 10 mM raffinose
Hapten: 50 mM GalNAc
Lectin Recognition of Glycans
Fucose-Binding in Complex-type N- and
O-glycans, and Glycosphingolipids
Hapten: 0.2 M Fuc
Hapten: 0.2 M Fuc
Hapten: 0.2 M a-methyl-Man
Hapten: 10 mM Fucose
Lectin Recognition of Glycans
N-Acetylglucosamine-Binding in Complex-type N- and
O-glycans, and Glycosphingolipids
[Hapten:10 mg/ml Chitotriose]
[Hapten: 0.1 M GlcNAc]
Lectin Recognition of Glycans
Sialic acid-Binding in Complex-type N- and
O-glycans, and Glycosphingolipids
[Hapten: 50 mM Lactose]
[Hapten: 50 mM Lactose]
Lectin Recognition of Glycans
Galactose- and N-acetylgalactosamine-Binding In O-glycans
[Hapten for all: 0.1 M GalNAc
[Hapten for all: 50 mM a-Methyl-GalNAc]
Serial Lectin Affinity Chromatography for Fractionation
and Purification of Complex Carbohydrates
Con A
Quantity of
Glycan
Fraction Number
LCA
L-PHA
L-PHA
LCA
SNA
SNA
Further Purification on Other Lectins, HPLC, etc.
Use of a lectin to assay a sialyltransferase
in an ELISA-type Method
Galb1-4GlcNAc-RCMP-NeuAc
a2-3-sialyltransferase
Step 1
CMP
NeuAca2-3Galb1-4GlcNAc-RAdd Biotinylated-MAL
Biotin-
BiotinCOLOR
Alk.Phos.Streptavidin-
Step 2
NeuAca2-3Galb1-4GlcNAc-R-
Add Alk.Phos.StreptavidinBiotin-
Step 3
NeuAca2-3Galb1-4GlcNAc-R-