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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 H3C OH B H3C 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 NH 2CO-; 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 RECOGNIT ION OF N-GL YCANS BY CONCANAVALIN A Mannose-Binding in N-Glycans a1,2 Man Man Bound Bound By a1,6 Man a1,2 a1,3 Man Man a1,2 a1,2 Man Man Man a1,6 b1,4 b1,4 Man GlcNAc GlcNAcb-Asn a1,3 Man a1,6 Man Man a1,3 b1,2 b1,4 Man Gal GlcNAc b1,4 Gal b1,2 Man GlcNAc b1,2 b1,4 Gal GlcNAc Man a1,6 b1,4 b1,4 Man GlcNAc GlcNAcb-Asn a1,3 a1,6 b1,4 b1,4 Man GlcNAc GlcNAcb-Asn a1,3 By Canavalia ensiformis Canavalia ensiformis lectin (Con A) lectin (Con A) (very strongly) (very 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 strongly) lectin (Con A) (weakly) 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 RECOGNITIONand OF N-GL YCANS CONT AINING TERMINAL Gal OR GalNAc RESIDUES O-glycans, Glycosphingolipids Bound By Bound By b1-4 Gal GlcNAc b1,6 b1-4 Gal GlcNAc-Man b1-4 a2-3 NeuAc Gal GlcNAc-Man GlcNAc b1,6 a1-3 b1-4 Gal GlcNAc-Man Gal b1-4 a2-3 NeuAc Gal GlcNAc-Man Erythrina cristagalli lectin (specific for Galb4GlcNAc-R) Ricinus communis agglutinin (RCA-I) RCA-I (strongly) Man-GlcNAc-GlcNAc-Asn (binds better to Galb4GlcNAc-R than To Galb3GlcNAc-R ) Hapten for both: 0.1 M lactose Man-GlcNAc-GlcNAc-Asn Griffonia simplicifolia agglutinin-I-B4 (GS-I-B4 ) Hapten: 10 mM raffinose a2-3 b1-4 NeuAc Gal GlcNAc b1,6 b1-4 GlcNAc-Man Gal GalNAc b1-4 GlcNAc-Man Man-GlcNAc-GlcNAc-Asn Wisteria floribunda agglutinin (WFA) Hapten: 50 mM GalNAc Dr. Cummings OF FUCOSYLA TED RECOGNITION Lectin Recognition of N-GLYCANS Glycans Fucose-Binding in Complex-type N- and O-glycans, and Glycosphingolipids Fuc a1,3 b1,4 GlcNAc-R Gal Bound BoundByBy Tetragonolobus purpureas agglutinin (TPA) (strongly), Aleuria aurantia lectin (AAL) (weakly) Hapten: 0.2 M Fuc Fuc a1,3 a2,3 b1,4 GlcNAc-R Gal NeuAc TPA (weakly), AAL (weakly) Hapten: 0.2 M Fuc Fuc R-GlcNAc-Man a1,6 a1,6 Man-GlcNAc-GlcNAc-Asn R-GlcNAc-Man a1,3 Lens culinaris agglutinin (Lentil lectin), Pisum sativum agglutinin (Pea lectin), AAL (strongly) Hapten: 0.2 M a-methyl-Man Fuc a1,2 b1,4 GlcNAc-R Gal Ulex europaeus agglutinin (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 ISOMERS OF POLYLACTOSAMINES Bound BoundByBy Galb1-4GlcNAcb1-3 Galb1-4GlcNAc b1-3Galb1-4GlcNAcb1-3Galb1-R n>2 Lycopersicon esculentum lectin (Tomato lectin), Solanum tuberosum lectin (potato lectin) and Galectins [Hapten:10 mg/ml Chitotriose] Galb1-4GlcNAc b1,6 Galb1-4GlcNAc b1-3Galb1-4GlcNAc b1-3Galb1-R Phytolacca americana mitogen (Pokeweed mitogen) and Triticum vulgaris agglutinin (Wheat germ agglutin -WGA) [Hapten: 0.1 M GlcNAc] Dr. Cummings Lectin Recognition of Glycans Sialic acid-Binding in Complex-type and RECOGNIT ION OF SIALNYLAT ED N-GLYCANS* O-glycans, and Glycosphingolipids Bound By By Bound NeuAc a2-3 b1-4 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: Many sialylated glycans are recognized weakly by WGA and Limulus polyphemus agglutinin; affinity is somewhat directly proportional to sialic acid content Dr. Cummings Lectin Recognition of Glycans Galactose- and N-acetylgalactosamine-Binding In O-glycans RECOGNIT ION OF O-GL Y CANS Bound By Bound By GalNAca1-R Vicia villosa agglutinin (VVA), Helix pomatia agglutinin (HPA), Wisteria floribunda agglutinin (weakly) [Hapten for all: 0.1 M GalNAc Gal b1,3 GalNAca1-R Arachis hypogaea agglutinin (peanut lectin - PNA), Artocarpus polyphem us 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