Transcript No Slide Title

ESSENTIALS OF GLYCOBIOLOGY
LECTURE 24
MAY 6, 2004
Richard D. Cummings, Ph.D.
University of Oklahoma Health Sciences Center
College of Medicine
Oklahoma Center for Medical Glycobiology
“THE PLANT LECTINS”
“Plant lectins have been to glycobiology as oligonucleotides have been to genetics except that plant lectins are practically free.”
Dr. Cummings
Outline
Historical Background On Plant Lectins
Classification And Sequence Of Plant Lectins
Toxicity Of Plant Lectins
Isolation Of Plant Lectins
Structure Of Plant Lectins
Functions Of Plant Lectins
Uses Of Plant Lectins
Dr. Cummings
“THE PLANT LECTINS”
 Definition of a Lectin (From the Latin verb legere
(meaning “to select”) (proposed by Boyd and Shapley in
1954)
 “A protein (other than an anti-carbohydrate antibody)
containing at least non-catalytic domain that specifically
recognizes and reversible binds to glycans”
 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.
Dr. Cummings
History of Plant Lectins
Date Investigators
1888
H. Stillmark
Discovery
Ricinus communis plant extract
has hemagglutinating properties
castor plant
1890
P. Ehrlich
Lectins (ricin and abrin) used as
immunogens in early
immunological studies
1908
K. Lansteiner &
H. Raubitsheck
Different hemagglutinating
properties found in extracts of
various plant seeds
1919
J. Sumner
Crystallization of Con A
1936
J. Sumner
Lectins bind sugar - Con A
precipitates glycogen
Dr. Cummings
History of Plant Lectins
Lens culinaris
(lentils)
Ricinus communis
(castor bean)
Dr. Cummings
History of Plant Lectins
Datura stramonium
(jimsonweed)
Aleuria aurantia
(Orange Cup)
Lycopersicum esculentum
(tomato)
Dr. Cummings
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
Dr. Cummings
History of Plant Lectins
Date Investigators
1960
P.C. Nowell
& J.C. Aub
Discovery
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
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
Dr. Cummings
SOME FAMILIES OF LECTINS
DISTINGUISHED BY 3º STRUCTURE
Lectin group
Structure of CRD
 R-type
b-trefoil (plants and animals)
(Ricin related)
 L-type
b-sandwich
 Hevein
 P58/ERGIC-53
b-sandwich
(Legume lectin-like)
 P-type
Unique b-rich structure
(Phosphomannose)
 M-type
Unique a-helical
(mannosidase-related
 C-type
Unique mixed a/b structure
(Ca2+-dependent)
 Galectins
b-sandwich
 I-type
Immunoglobulin superfamily
# of Residues
~125
~230
~43
~220
~130
~500
~115
~125
~120
Dr. Cummings
Classification of Plant Lectins Based
on Protein Folding Domains
1.
2.
3.
4.
5.
b -Trefoil lectin
Legume
Agglutinin with hevein domain
Monocot
b-Prism
Dr. Cummings
Primary Structural Motifs in Leguminous Plant Lectins
N-TERMINI
A-E-L-F-F-N-F-Q-T-F-N-A-A-N- Lima bean lectin Phaseolus limensis
A-E-T-V-S-F-S-W-N-K-F-V-P-K-Q- SBA - Soybean agglutinin (Glycine max)
F
E Y
L
L
S CONSERVED MOTIF IN
- I -x- D - W -V-x- I -G- F - L T C-TERMINAL DOMAIN
Q K
V
V
R
Red = invariant
residues
-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
T
- I - -V- Q - L -D- S - METAL BINDING
A
T
V I
V
SITES
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
(Top) Comparison of the amino termini of two examples of leguminous lectins, mature lima bean lectin and soybean agglutinin.
(Middle) A conserved motif in the carboxy-terminal domains of leguminous lectins. Invariant residues are indicated by boldfaced
letters and con-served residues are shown in parentheses. Nonconserved amino acids are indicated by -x-. The sequences of lima bean
lectin and soybean agglutinin are compared. Identical residues are boxed. (Bottom ) Consensus pattern of the metal-binding domain in
the carboxyl terminus of leguminous lectins.
Dr. Cummings
Properties of Some Plant Lectins
Class
Subunits
Binding
Sites per
Subunit
25-30
2 or 4
1
2
2
Monosaccharide Subunit MW
Specificity
(kDa)
L-Type
Diverse
Grains
Primarily Amino
Sugars
(GlcNAc/NeuAc)
~18
Glycosylation
-S-SBonds
Metals
(HeveinType)
Class
L-type
Variable
No
Mn2+,
Ca2+
Grains
Variable
Yes
No
(Hevein-Type)
Dr. Cummings
Classification of Plant Lectins
1. b-trefoil lectin (R-type)
Closed barrel with a hairpin triplet; internal duplication; internal pseudo
threefold symmetry; three lobes arranged as a b-trefoil around a 3-fold axis
abrin-a
amaranthin
castor bean ricin B
ebulin
Misteltoe lectin
TKL-1
Dr. Cummings
Structures of R-type Lectins
Comparisons between Cys-MR (R-type domain in the mannose
receptor) and other b-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)
Closed barrel with a hairpin triplet; internal duplication; internal pseudo
threefold symmetry; three lobes arranged as a b-trefoil around a 3-fold axis
Dr. Cummings
Crystallographic structures of ricin (A) and Shiga toxin (B)
• This binding by the B-chain is a requirement
for internalization and eventual translocation
of the A-chain into 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).
Shiga toxin (Stx) from Shigella dysenteriae serotype
• The cytosolic target of ricin and Shiga toxin
is the 28S RNA of the 60S ribosomal subunit
(Endo et al., 1987), where depurination and
inactivation results. Ricin A-chain cleaves the
N-glycosidic bond at A-4324 in 28 S rRNA
when intact rat ribosomes are the substrate.
Cleavage occurs at a concentration of the
toxin of 1 x 10-10 M, and specificity for this
single residue is retained when the
concentration is as high as 3 x 10-7M.
• Reduction of the disulfide bond connecting
the A- and B-chains of ricin is required for
optimal enzymatic activity.
Dr. Cummings
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
Dr. Cummings
Crystal Structure of the b-Trefoil lectin Abrin from Abrus precatorius
B chain
A chain
Tahirov, et al, (1995) J. Mol. Biol., 250, 354-367
Dr. Cummings
Classification of Plant Lectins
2. Legume lectin (L-type)
canonical twelve-stranded beta-sandwich structure
Canavalia brasiliens
concanavalin A
Cratylia mollis
Dioclea grandiflora DGL
Dioclea guianensis lectin
Dolichos biflorus DB58
Dolichos biflorus DBL
Dolichos lablab FRIL
Erythrina corallodendron EcorL
Erythrina cristallogali ECL
favin
Griffonia simplicifolia GS-I
Griffonia simplicifolia GS-IV
Lathyrus ochrus LOL-1
Lathyrus ochrus LOL-2
Lentil LCL
lima bean LBL
Maackia amurensis MAL
Pea PSL
Peanut PNA
Phaseolus vulgaris PHA-L
Pterocarpus angolensis
Robinia pseudoacacia bark lectin I
Soybean SBA
Ulex europaeus UEA-1
Ulex europaeus UEA-2
Vicia villosa VVL-B4
winged bean agglutinin I
winged bean agglutinin II
Dr. Cummings
Evolution of L-type Lectins
Human
ERGIC
VIP
Animals
L-type CRD
Yeast
Plants
EMP47
ERGIC
VIP
Drosophila ERGIC
VIP
C. elegans
ERGIC
VIP
Seed
Lectins
Dr. Cummings
Canavalia ensiformis (Con A) (2.35 Å) Complexed with aMan1-3(aMan1-6)Man
Trimannose
Homotetramer;
each subunit
colored
differently
Naismith, J.H., Field, R.A., Structural basis of trimannoside recognition
by concanavalin A:, J. Biol. Chem., 271, 972-976, 1996
Dr. Cummings
Maackia amurensis Lectin Crystallized with Sialyl Lactose
Ca2+ and Mn2+ ions are displayed as green
and magenta spheres, respectively, and water
molecules as small blue spheres.
Imberty et al, J. Biol. Chem., 275, 17541-17548
Dr. Cummings
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
Dr. Cummings
The crystal structure of the L-type lectin from Canavalia brasiliensis
Sanz-Aparicio, et al, (1997) FEBS Letters, 405, 114-118
Dr. Cummings
Similarities in Protein Folding Between
Galectins and Legume L-type Lectins
Con A Dimer
Bovine Galectin-1 Dimer
Dr. Cummings
Classification of Plant Lectins
3. Agglutinin with hevein domain
Hevein, a wound-induced protein found in the latex of Hevea brasiliensis (rubber tree).
A conserved domain of 43 amino acids found in several plant and fungal proteins that
have a common binding specificity for oligosaccharides of N-acetylglucosamine.
Hevein
Pokeweed lectin
Urtica dioica UDA
Wheat germ WGA-1
Wheat germ WGA-2
Wheat germ WGA-3
Hevein
Crystal structure of the Urtica dioica lectin
complexed with chitotetraose. From Saul et
al (2000) Structure 8, 593-603
Dr. Cummings
Classification of Plant Lectins
4. a-D-mannose-specific plant lectin (monocot lectin)
all contain a tertiary structure consisting of three sequential beta-sheet subdomains
(I, II and III) related by a pseudo 3-fold axis of symmetry.
amaryllis
Bluebell SCA-FET
Bluebell SCA-MAN
daffodil amaryllidaceae
garlic bulbs lectin
snowdrop lectin
Garlic bulb lectin (Allium sativum) complexed with a-Man (from
Ramachandraiah, et al (2002) Acta Crystallogr. D. 58, 414-420
(note: each subunit has 3 binding sites for mannose)
Dr. Cummings
Classification of Plant Lectins
5. b-prism plant lectin
(also called Jacalin or Jacalin-like proteins)
consists of 3 4-stranded sheets; strands are perpendicular to the 3-fold axis
duplication: consists of two domains of this fold
Artocarpin (Artocarpus integrifolia)
Calsepa
heltuba
jacalin
Maclura pomifera MPA
Dr. Cummings
Protein Folding in b-prism-type Lectins
consists of 3 4-stranded sheets; strands are perpendicular to the 3-fold axis
duplication: consists of two domains of this fold
Crystal structure of artocarpin lectin from the jack fruit
(Artocarpus integrifolia) (left - monomer; right - tetramer)
Dr. Cummings
Crystal structure of the b-Prism Lectin
from Helianthus tuberosus Complexed with aMan1-3Man
consists of 3 4-stranded sheets; strands are perpendicular to the 3-fold axis
duplication: consists of two domains of this fold
Bourne, et al, (1999) Structure, 7, 1473-1482
Dr. Cummings
Because of their multivalency and oligomeric
structures, many plant lectin can cross-linking can
precipitate glycoproteins and agglutinate cells
Dr. Cummings
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 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.
Dr. Cummings
Ricin Biosynthesis
• Ricin and RCA are produced by endosperm cells of maturing seeds.
• Once synthesized they are stored in protein bodies, a vacuolar organelle.
• After seed germination, the proteins are completely degraded within days.
• Ricin is synthesized as a prepropolypeptide containing both the A and B chains and an Nterminal signal sequence targeting it to the ER.
• The prepropolypeptide is N-glycosylated co-translationally and the signal sequence removed.
• Proricin is formed, acquiring a single disulfide bond that will eventually link the A and B
chains, and acquires complex N-glycans.
• Within the protein bodies, proricin is processed by a endopeptidase releasing the A and B
chain, which remain associated by disulfide bonds.
• The A and B chains remain connected by the disulfide linkage.
• This complex processing pathway for ricin presumably prevents the A chain from disrupting
protein biosyntheis within the plant itself.
• No active ligands have been found in endosperm for ricin, hence ricin is considered
packaged and inert until released from the storage granules, either by normal germination
processes or ingestion by predators.
Dr. Cummings
Typical Leguminous Plant N-Glycan Structures
Dr. Cummings
Biological Functions of Plant Lectins
 Seed storage proteins (plant lectins are also called
vegetative storage proteins or VSPs)
 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
Dr. Cummings
Plant Lectin Function in Nitrogen Fixation/Rhizobial Infection
•
•
•
•
•
•
•
•
Plant nodulation proceeds by nodulating bacteria (rhizobia) interacting with root hairs.
Nodulation requires that differentiated root cells be re-differentiated into a primordium, that is
required for root nodulation.
When rhizobia interact and colonize the root hairs, they induce morphological changes, and gene
expression in the dermis.
Nodulation by rhizobia caused deformation of root hairs and reinitiates root hair growth, but now
it is inward instead of outward.
The nodulating bacteria produce Nod factor signals (see the lipo-chito-oligosaccharides (LCOs)
on next slide), sensed by the plant, and resulting in nodule formation.
The sensing of the LCOs may involve plant lectins.
The roots of the legume Dolichos biflorus contain a lectin/nucleotide phosphohydrolase (DbLNP) that binds to the LCOs 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).
Additional Ref: Localization of a lipo-chito oligosaccharides (LCOs), or Nod factors and Nod factorbinding 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
Dr. Cummings
Nodulation in Legumes
Schematic Drawing of Early
Steps in Root Nodule Formation
in Medicago truncatula Induced
by Sinorhizobium meliloti.
Symbiotic bacteria
attach to root hair
and invade root
Root cross-section
The major Nod factor produced by
Sinorhizobium meliloti contains four
glucosamine units, an acyl chain of 16 Catoms in length with two unsaturated
bonds (determined by NodE and NodF),
an acetyl group at the non-reducing
terminal sugar residue (determined by
NodL), and a sulfate group at the
reducing terminal sugar residue
(determined by NodH, NodP and NodQ)
OH
OR
HO
HO
OH
H3 C
OH
B
H3 C
OH
RO
OH
NAc
A
HO
NHFatty Acid
NAc
HO
C
D
OH
_
±SO3 In some LCOs
F
E
HO
NHFatty Acid
OH
G
OH
NHHFatty Acid
_
±SO3 In some LCOs
Structure of lipo-chito 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
Dr. Cummings
Some 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
 Mitogenesis of cells
 Mapping neuronal pathways
 Purification and characterization of glycoconjugates
 Assays of glycosyltransferases and glycosidases
 Defining glycosylation status of target glycoconjugates
Dr. Cummings
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
Dr. Cummings
Serial Lectin Affinity Chromatography (SLAC) for Fractionation and
Purification of Complex Carbohydrates
Con A
Quantity of Glycan
A
Elution Conditions at arrows:
A - 10 mM a-methylGlc;
B - 100 mM a-methylMan;
C - 50 mM lactose
B
High mannose- and hybrid-type N-glycans
Biantennary complex-type N-glycans
Tri- Hexaantennary complex-type N-glycans
and all O-glycans
Fraction
LCA
LCA
B
L-PHA
B
L-PHA
SNA
C
Triantennary complex-type N-glycans with
core a6-fucose
Biantennary complex-type N-glycans
with core a6-fucose
SNA
C
Further Purification on Other Lectins, HPLC, etc.
Dr. Cummings
Lectin Recognition of Glycans
RECOGNITION OF N-G LYCANS BY CONCANAVALIN A
Mannose-Binding in N-Glycans
a1,2
Man
Man
Bound
Bound
By
a1,6
Man
a1,6
b1,4
b1,4
Man
GlcNAc
GlcNAc
b-Asn
a1,2
a1,3
Man
Man
a1,2
a1,2
a1,3
Man
Man
Man
Man a1,6
Man
Man a1,3
b1,2
b1,4
Man
Gal
GlcNAc
b1,4
Gal
b1,4
Gal
b1,2
Man
GlcNAc
a1,6
b1,4
b1,4
Man
GlcNAc
GlcNAc
b-Asn
a1,3
a1,6
b1,4
b1,4
Man
GlcNAc
GlcNAc
b-Asn
b1,2
a1,3
Man
GlcNAc
By
Canavalia ensiformis
Canavalia
lectinensiformis
(Con A)
lectin (Con A)
strongly)
(v(very
ery strongly)
Hapten: 0.5 M
a-Methyl Man
Canavalia ensiformis
lectinensiformis
(Con A)
Canavalia
lectin
A)
(very(Con
strongly)
(strongly)
Hapten: 0.1 M
a-Methyl Man
Canavalia ensiformis
lectin (Con A)
Canavalia ensiformis
(very(Con
strongly)
lectin
A)
(w
eakly)
Hapten: 0.1 M
a-Methyl Glc
Dr. Cummings
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
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
Dr. Cummings
Lectin Recognition of Glycans
Galactose-Binding in Complex-type N- and
RECOGNITION
OFGlycosphingolipids
N-GL
YCANS CONTAINING TERMI NALGal OR GalNAc RESI DUES
O-glycans,
and
Bound By
By
Bound
b1-4
Gal
GlcNAc
b1,6
Erythrina cristagalli lectin
b1-4
(specific for Galb4GlcNAc-R)
Gal
GlcNAc-Man
Ricinus communis
agglutinin
RCA-I (str
ongl y)(RCA-I)
b1-4
Man-GlcNAc-GlcNAc-Asn
a2-3
(binds better to Galb4GlcNAc-R than
NeuAc
Gal
GlcNAc-Man
To Galb3GlcNAc-R )
GlcNAc
Hapten
b1,6
a1-3
b1-4
Gal
GlcNAc-Man
Gal
b1-4
a2-3
Man-GlcNAc-GlcNAc-Asn
NeuAc
Gal
GlcNAc-Man
for both: 0.1 M lactose
Gri ffonia si mpl icifolia
aggl utini n-I-B
4 (GS-I-B4 )
Hapten: 10 mM raffinose
a2-3
b1-4
NeuAc
Gal
GlcNAc
b1,6
b1-4
GlcNAc-Man
Gal
b1-4
GalNAc
GlcNAc-Man
Man-GlcNAc-GlcNAc-Asn
Wister ia flori bunda
aggl utini n (WF
A)
Hapten: 50 mM GalNAc
Dr. Cummings
N-GLYCANS
TED
TION OF FUCOSYLA
RECOGNIRecognition
Lectin
of Glycans
Fucose-Binding in Complex-type N- and
O-glycans, and Glycosphingolipids
Bound
BoundBy
By
Tetr agonol obus
purpureasaggluti ni n
(TPA) (strongly),
Aleuri a aur antial ectin
(AAL) (weakly)
Fuc
a1,3
b1,4
Gl cNAc-R
Gal
Hapten: 0.2 M Fuc
NeuAc
a2,3
b1,4
Gal
Fuc
a1,3
Gl cNAc-R
TPA (weakly),
AAL (weakly)
Hapten: 0.2 M Fuc
Fuc
a1,6
a1,6
Man-GlcNAc-Gl cNAc-Asn
R-Gl cNAc-Man a1,3
R-Gl cNAc-Man
Lens culi nar is
aggl utinin (Lentil lecti n),
Pisum sativum
aggl utinin (Pea lectin),
AAL (strongly)
Hapten: 0.2 M a-methyl-Man
Fuc
a1,2
b1,4
Gl cNAc-R
Gal
Ulex europaeus
aggl utinin (UEA-I),
AAL (weakly)
Hapten: 10 mM Fucose
Dr. Cummings
Lectin Recognition of Glycans
N-Acetylglucosamine-Binding in Complex-type N- and
O-glycans, and Glycosphingolipids
RECOGNITION OF STRUCTURAL
I SOMERS OF POL
YLACTOSAMI NES
Bound
BoundBy
By
Galb1-4GlcNAcb1-3 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-R
n>2
Lycoper sicon esculentum
lecti n (Tomato l ecti n),
Solanum tuberosum
l ectin
(potato lectin) and
Galectins
Galb1-4GlcNAc
b1,6
Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-R
[Hapten:10 mg/ml Chitotriose]
Phytolacca amer icanamitogen
(Pokeweed mitogen)
and
Tr iti cum vul gar isaggl utinin
(W heat ger m aggl utin -WGA)
[Hapten: 0.1 M GlcNAc]
Dr. Cummings
Lectin Recognition of Glycans
Sialic acid-Binding
in Complex-type
Nand N-GLYCANS*
RECOGNITION
OF SIAL
YLATED
O-glycans, and Glycosphingolipids
BoundBy
By
Bound
b1-4
NeuAc a2-3 Gal
GlcNAc
-R
Maackia amurensis
leukoagglutinin (MAL)
[Hapten: 50 mM Lactose]
NeuAc
a2-6
Gal(or GalNAc)
-R
Sambucus nigra
agglutinin (SNA)
[Hapten: 50 mM Lactose]
* Note: M any sialylated glycans are recognized w eakly
by WGA and Limulus polyphemusagglutinin; affinity is
somew hat directly proportional to sialic acid content
Dr. Cummings
Lectin Recognition of Glycans
Galactose- and N-acetylgalactosamine-Binding
In O-glycans
RECOGNITION OF O-GLYCANS
Bound By
Bound
By
GalNAca1-R
Vicia villosa
agglutinin (VVA),
Helix pom atia
agglutinin (HPA),
Wisteria floribunda
agglutinin (w eakly)
[Hapten for all: 0.1 M GalNAc
Gal
b1,3
GalNAc
a1-R
Arachis hypogaea
agglutinin (peanut
lectin - PNA),
Artocarpus
polyphemus lectin
(Jacalin lectin)
[Hapten for all: 50 mM a-Methyl-GalNAc]
Dr. Cummings
Use of a lectin to assay a sialyltransferase
in an ELISA-type Method
Galb1-4GlcNAc-RCMP-NeuAc
a2-6-sialyltransferase
Step 1
CMP
NeuAca2-6Galb1-4GlcNAc-RAdd Biotinylated-SNA
Biotin-
SNA
Step 2
Biotin- SNA NeuAca2-6Galb1-4GlcNAc-RCOLOR
Alk.Phos.Streptavidin-
Add Alk.Phos.StreptavidinBiotin- SNA
Step 3
NeuAca2-6Galb1-4GlcNAc-RDr. Cummings