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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.

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Presentation on theme: "ESSENTIALS OF GLYCOBIOLOGY LECTURE 24 MAY 6, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma."— Presentation transcript:

1 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

2  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 Outline Dr. Cummings

3  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. “THE PLANT LECTINS” Dr. Cummings

4 1888H. Stillmark Ricinus communis plant extract has hemagglutinating properties 1890 P. Ehrlich Lectins (ricin and abrin) used as immunogens in early immunological studies 1908 K. Lansteiner &Different hemagglutinating H. Raubitsheck properties found in extracts of various plant seeds 1919 J. SumnerCrystallization of Con A 1936J. SumnerLectins bind sugar - Con A precipitates glycogen DateInvestigatorsDiscovery History of Plant Lectins castor plant Dr. Cummings

5 Lens culinaris (lentils) Ricinus communis (castor bean) History of Plant Lectins Dr. Cummings

6 Aleuria aurantia (Orange Cup) Lycopersicum esculentum (tomato) Datura stramonium (jimsonweed) History of Plant Lectins Dr. Cummings

7 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. Morganprove that blood group antigens are sugars and to deduce the structures of the antigens 1954 W. Boyd &The name lectin is proposed to E. Shyleigh replace hemagglutinin DateInvestigatorsDiscovery History of Plant Lectins Dr. Cummings

8 1960P.C. NowellRed kidney bean lectin P. vulgaris & J.C. Aub mitogenic for resting lymphocytes 1960’s M. Burger Lectins preferentially 1970’s G. Nicolson agglutinate some animal tumor cells 1980’s Kornfeld(s)Use of immobilized lectins to analyze Osawaanimal glycoconjugates Kobata 1980’s D. KabelitzDiscovery that plant lectins induce 1990’s D.J. Geeapoptosis K. Schweizer DateInvestigatorsDiscovery History of Plant Lectins Dr. Cummings

9 Lectin groupStructure of CRD # of Residues  R-type  -trefoil (plants and animals)~125 (Ricin related)  L-type  -sandwich~230  Hevein ~43  P58/ERGIC-53  -sandwich ~220 (Legume lectin-like)  P-typeUnique  -rich structure~130 (Phosphomannose)  M-typeUnique  -helical~500 (mannosidase-related  C-typeUnique mixed  /  structure ~115 (Ca 2+ -dependent)  Galectins  -sandwich~125  I-typeImmunoglobulin superfamily~120 SOME FAMILIES OF LECTINS DISTINGUISHED BY 3º STRUCTURE Dr. Cummings

10 1.  -Trefoil lectin 2. Legume 3. Agglutinin with hevein domain 4. Monocot  -Prism Classification of Plant Lectins Based on Protein Folding Domains Dr. Cummings

11 METAL BINDING SITES - - -V- - -D- - LIVLIV STAGSTAG EQVEQV FLIFLI STST -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 Plant Lectins Red = invariant residues (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. - -x- - -V-x- -G- - - LIVLIV EDQEDQ FYWKRFYWKR LIVLIV FLFL STST Dr. Cummings

12 L-Type Grains (Hevein- Type) Diverse Primarily Amino Sugars (GlcNAc/NeuAc) ~18 2 or Class Monosaccharide Specificity Subunit MW (kDa) Subunits Binding Sites per Subunit Properties of Some Plant Lectins L-type Grains (Hevein-Type) Variable No Yes Mn 2+, Ca 2+ No Class Glycosylation -S-S- Bonds Metals Dr. Cummings

13 1.  -trefoil lectin (R-type) abrin-a amaranthin castor bean ricin B ebulin Misteltoe lectin TKL-1 Classification of Plant Lectins Closed barrel with a hairpin triplet; internal duplication; internal pseudo threefold symmetry; three lobes arranged as a  -trefoil around a 3-fold axis Dr. Cummings

14 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: ) Structures of R-type Lectins Dr. Cummings Closed barrel with a hairpin triplet; internal duplication; internal pseudo threefold symmetry; three lobes arranged as a  -trefoil around a 3-fold axis

15 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). 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 M, and specificity for this single residue is retained when the concentration is as high as 3 x M. Reduction of the disulfide bond connecting the A- and B-chains of ricin is required for optimal enzymatic activity. Crystallographic structures of ricin (A) and Shiga toxin (B) Dr. Cummings Shiga toxin (Stx) from Shigella dysenteriae serotype

16 RRRRRRRRR Drosophila Lectins Bacterial Lectins Bacterial Hydrolases Ricin/Plant Toxins GalNAc Transferases Mannose Receptor Family RCCCC CCCC R R-type CRD GalNAcT Domain Hydrolase Domain C C-type CRD TM domain Fibronectin domain Ricin-type R-type Lectins -  -trefoil proteins Dr. Cummings

17 Tahirov, et al, (1995) J. Mol. Biol., 250, Crystal Structure of the  -Trefoil lectin Abrin from Abrus precatorius Dr. Cummings B chain A chain

18 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 2. Legume lectin (L-type) Classification of Plant Lectins canonical twelve-stranded beta-sandwich structure Dr. Cummings

19 L-type CRD Plants Animals Yeast EMP47 ERGIC VIP C. elegans Drosophila ERGIC VIP Human ERGIC VIP ERGIC VIP Seed Lectins Evolution of L-type Lectins Dr. Cummings

20 Naismith, J.H., Field, R.A., Structural basis of trimannoside recognition by concanavalin A:, J. Biol. Chem., 271, , 1996 Canavalia ensiformis (Con A) (2.35 Å) Complexed with  Man1-3(  Man1-6)Man Dr. Cummings Homotetramer; each subunit colored differently Trimannose

21 Maackia amurensis Lectin Crystallized with Sialyl Lactose Ca 2+ and Mn 2+ ions are displayed as green and magenta spheres, respectively, and water molecules as small blue spheres. Imberty et al, J. Biol. Chem., 275, Dr. Cummings

22 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 Mn 2+ (green) and Ca 2+ (yellow) of the canonical (S1 and S2) metal-binding site are shown as spheres. The secondary sub-sites for the Ca 2+ /Cd 2+ (S3) and Mn 2+ (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 Dr. Cummings

23 Sanz-Aparicio, et al, (1997) FEBS Letters, 405, The crystal structure of the L-type lectin from Canavalia brasiliensis Dr. Cummings

24 Bovine Galectin-1 Dimer Con A Dimer Similarities in Protein Folding Between Galectins and Legume L-type Lectins Dr. Cummings

25 3. Agglutinin with hevein domain Hevein Pokeweed lectin Urtica dioica UDA Wheat germ WGA-1 Wheat germ WGA-2 Wheat germ WGA-3 Classification of Plant Lectins 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. Crystal structure of the Urtica dioica lectin complexed with chitotetraose. From Saul et al (2000) Structure 8, Hevein Dr. Cummings

26 4.  -D-mannose-specific plant lectin (monocot lectin) amaryllis Bluebell SCA-FET Bluebell SCA-MAN daffodil amaryllidaceae garlic bulbs lectin snowdrop lectin Classification of Plant Lectins Garlic bulb lectin (Allium sativum) complexed with  -Man (from Ramachandraiah, et al (2002) Acta Crystallogr. D. 58, (note: each subunit has 3 binding sites for mannose) 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. Dr. Cummings

27 5.  -prism plant lectin (also called Jacalin or Jacalin-like proteins) Artocarpin (Artocarpus integrifolia) Calsepa heltuba jacalin Maclura pomifera MPA Classification of Plant Lectins consists of 3 4-stranded sheets; strands are perpendicular to the 3-fold axis duplication: consists of two domains of this fold Dr. Cummings

28 Crystal structure of artocarpin lectin from the jack fruit (Artocarpus integrifolia) (left - monomer; right - tetramer) Protein Folding in  -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 Dr. Cummings

29 Bourne, et al, (1999) Structure, 7, Crystal structure of the  -Prism Lectin from Helianthus tuberosus Complexed with  Man1-3Man consists of 3 4-stranded sheets; strands are perpendicular to the 3-fold axis duplication: consists of two domains of this fold Dr. Cummings

30 Because of their multivalency and oligomeric structures, many plant lectin can cross-linking can precipitate glycoproteins and agglutinate cells Dr. Cummings

31  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 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 Dr. Cummings

32 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 N- terminal 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. Ricin Biosynthesis Dr. Cummings

33 Typical Leguminous Plant N-Glycan Structures Dr. Cummings

34  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 Biological Functions of Plant Lectins Dr. Cummings

35 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 (Db- LNP) 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 factor- binding protein in legume roots and factors influencing its distribution and expression. Plant Physiol 124(3): ). 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 Dr. Cummings

36 Schematic Drawing of Early Steps in Root Nodule Formation in Medicago truncatula Induced by Sinorhizobium meliloti. Nodulation in Legumes Symbiotic bacteria attach to root hair and invade root Dr. Cummings The major Nod factor produced by Sinorhizobium meliloti contains four glucosamine units, an acyl chain of 16 C- atoms 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) Root cross-section

37  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 Dr. Cummings

38 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) Dr. Cummings

39 Con A LCA L-PHA Fraction SNA Further Purification on Other Lectins, HPLC, etc. SNA Quantity of Glycan Serial Lectin Affinity Chromatography (SLAC) for Fractionation and Purification of Complex Carbohydrates High mannose- and hybrid-type N-glycans Biantennary complex-type N-glycans Tri- Hexaantennary complex-type N-glycans and all O-glycans Elution Conditions at arrows: A - 10 mM  -methylGlc; B mM  -methylMan; C - 50 mM lactose A B Triantennary complex-type N-glycans with core  6-fucose Biantennary complex-type N-glycans with core  6-fucose B B C C Dr. Cummings

40 Lectin Recognition of Glycans Mannose-Binding in N-Glycans Canavalia ensiformis lectin (Con A) (very strongly) Hapten: 0.5 M  -Methyl Man Canavalia ensiformis lectin (Con A) (very strongly) Hapten: 0.1 M  -Methyl Man Canavalia ensiformis lectin (Con A) (very strongly) Hapten: 0.1 M  -Methyl Glc Bound By Dr. Cummings

41 Phaseolus vulgaris leukoagglutinin (L 4 -PHA) Hapten: 0.4 M GalNAc Datura stramonium agglutinin (DSA) (weakly) Hapten: 10 mg/ml Chitotriose Phaseolus vulgaris erythroagglutinin (E 4 -PHA) Hapten: 0.4 M GalNAc Man-GlcNAc-GlcNAc-Asn Gal GlcNAc Man  1,4 Gal GlcNAc Man-GlcNAc-GlcNAc-Asn  1,6 Man-GlcNAc-GlcNAc-Asn GlcNAc  1,4 Bound By  1,4  1,2 Gal GlcNAc Man  1,4  1,2  1,4 Gal GlcNAc  1,4 Gal GlcNAc Man  1,2 Gal GlcNAc Man  1,4  1,2 Gal GlcNAc Man  1,4  1,2 Gal GlcNAc Man  1,4  1,2 Lectin Recognition of Glycans  1,4 Galactose-Binding in Complex-type N-glycans Bound By Dr. Cummings

42 Hapten for both: 0.1 M lactose Hapten: 10 mM raffinose Hapten: 50 mM GalNAc Erythrina cristagalli lectin (specific for Gal  4GlcNAc-R) Ricinus communis agglutinin (RCA-I) (binds better to Gal  4GlcNAc-R than To Gal  3GlcNAc-R ) Lectin Recognition of Glycans Galactose-Binding in Complex-type N- and O-glycans, and Glycosphingolipids Bound By Dr. Cummings

43 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 Bound By Dr. Cummings

44 [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 Bound By Dr. Cummings

45 [Hapten: 50 mM Lactose] Lectin Recognition of Glycans Sialic acid-Binding in Complex-type N- and O-glycans, and Glycosphingolipids Bound By Dr. Cummings

46 [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 Bound By Dr. Cummings

47 Add Alk.Phos.- Streptavidin- Alk.Phos.- Streptavidin- Biotin- Use of a lectin to assay a sialyltransferase CMP Gal  1-4GlcNAc-R- CMP-NeuAc NeuAc  2-6Gal  1-4GlcNAc-R- Add Biotinylated-SNA Step 1 Step 2 Step 3  2-6-sialyltransferase in an ELISA-type Method COLOR Dr. Cummings SNA

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