Biomedical subjects

I J Goldstein

Publications and source records attributed to I J Goldstein.

At least 19 recordsLinked to original sources

PubMed1992

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Peptide ligands for the carbohydrate-binding protein concanavalin A (Con A) have been identified by screening a large, diverse peptide library expressed on the surface of filamentous phage. A dodecapeptide containing the consensus sequence Tyr-Pro-Tyr was found to bind Con A with an affinity (dissociation constant, Kd) of 46 microM, comparable to that of a known carbohydrate ligand, methyl alpha-D-mannopyranoside (Kd of 89 microM). In addition the peptide inhibited precipitation of the alpha-glucan dextran 1355 by Con A. Given the complexity of oligosaccharide synthesis, the prospect of finding peptides that competitively inhibit carbohydrate-specific receptors may simplify the development of new therapeutic agents.

Amino Acid Sequence↗

PubMed1992

A family of concanavalin A-binding peptides from a hexapeptide epitope library.

The lectin concanavalin A (Con A) binds methyl alpha-D-mannopyranoside (Me alpha Man) as well as alpha-D-mannosyl groups at the nonreducing terminus of oligosaccharides. Ligand peptides that mimic the binding of Me alpha Man to Con A were identified from screening an epitope library composed of filamentous phage displaying random hexapeptides. A consensus sequence was identified among affinity-purified phage; Con A binds phage bearing this sequence and is inhibited from doing so by Me alpha Man. When tested for binding against a panel of lectins, phage bearing this sequence bind only weakly to a closely related D-mannose-binding lectin, indicating that binding to Con A is highly selective. A synthetic peptide bearing the consensus sequence blocks the precipitation of Con A by dextran with an inhibition strength equivalent to that of methyl alpha-D-glucopyranoside. These results demonstrate that the specificity of Con A is not limited to carbohydrates and that highly selective sugar-mimics for lectins of plant, animal, or bacterial origin may be identified from epitope libraries.

Amino Acid Sequence↗

PubMed1992

The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.

Amino Acid Sequence↗

PubMed1992

Interaction of linear manno-oligosaccharides with three mannose-specific bulb lectins. Comparison with mannose/glucose-binding lectins.

Three new mannose-binding lectins, isolated from daffodil (NPA), amaryllis (HHA), and snowdrop (GNA) bulbs, are capable of precipitating with a linear mannopentaose (Man alpha 1-3Man alpha 1-3Man alpha 1-3Man alpha 1-2Man). NPA and HHA reacted strongly with the mannopentaose whereas GNA gave a precipitate only at concentrations greater than 500 microM. A phosphate group at C-6 of the nonreducing terminal mannosyl group prevented precipitation in all three cases. The reduced (NaBH4) mannopentaose, Man4Man-ol, did not precipitate with GNA or NPA, but was active with HHA. This activity was lost when Man4Man-ol was converted (NaIO4 then NaBH4; mild acid hydrolysis of the reduced product) into trisaccharide derivatives. With alpha-D-Manp-OMe the three lectins gave UV difference spectra having large positive peaks at 292-293 and 283-284 nm, and a small positive peak at 275 nm, characteristic of tryptophanyl and tyrosyl residues. The association constants for the interaction with alpha-D-Manp-OMe were very low (NPA, 86; HHA, 66; and GNA, 41 M-1), but the lectins bound methyl (1----3)-alpha-mannobioside with increased affinity (K for NPA 540, for HHA 2400, and for GNA 200 M-1). The bulb lectins lack binding sites for hydrophobic ligands, as judged by their failure to interact with the fluorescent probes 8-anilino-1-napthalenesulfonic acid (ANS) and 6-p-toluidino-2-naphthalenesulfonic acid (TNS).

Carbohydrate Sequence↗

PubMed1992

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.

Allium↗

PubMed1992

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.

Animals↗

PubMed1992

Lectin reactivities as intermediate biomarkers in premalignant colorectal epithelium.

Normal colonic epithelial cells undergo maturation as they traverse the crypt to the lumenal surface. The binding of lectins to goblet cell mucins and other glycoconjugates changes as the cells migrate and differentiate. Additional stepwise modifications in glycoconjugate expression occur in premalignant and malignant neoplasms that may be detected by lectin binding studies. The lectins Dolichos biflorus agglutinin (DBA) and soybean agglutinin (SBA) have been developed as markers of differentiation in normal-appearing colonic epithelium. Using a quantitative biometric system to score tissues, reduced levels of lectin binding have been found in rectal tissue from patients with familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer. The lectin Amaranthus caudatus agglutinin (ACA) binds to a cytoplasmic glycoconjugate expressed at the base of the colonic crypt and serves as a possible proliferation marker in the distal, but not proximal, colon. ACA binding increases in tandem with increased levels of proliferation (using BrdU incorporation) in neoplastic tissues. Binding by the peanut lectin (PNA) occurs late in the adenoma-to-carcinoma sequence--in larger adenomas and in cancers--and serves as a marker of advancing neoplasia. Lectins identify the stepwise changes that occur during normal differentiation, proliferation and in advancing neoplasia. By selecting the appropriate probe, biomarkers may be developed for early, intermediate, and late events in colorectal cancer.

Biomarkers↗

PubMed1992

Synthesis of high-affinity, hydrophobic monosaccharide derivatives and study of their interaction with concanavalin A, the pea, the lentil, and fava bean lectins.

Concanavalin A (Con A) and agglutinins from the pea (PSA), lentil (LCH), and fava bean (VFA) constitute a group of D-mannose/D-glucose binding legume lectins. In addition to their sugar binding specificity, these lectins also contain sites that bind hydrophobic ligands. The present study explores a class of nonpolar binding sites reportedly present adjacent to the carbohydrate binding site in PSA, LCH, and VFA. A series of 2-O- and 3-O-substituted nitrobenzoyl and nitrobenzyl derivatives of methyl alpha-D-glucopyranoside and methyl alpha-D-mannopyranoside were synthesized. Evaluation of their binding to Con A, PSA, LCH, and VFA was carried out by the technique of hapten inhibition of precipitation reaction. The hapten inhibition assay results reveal that the presence of a methyl or methylene group at the O-2 or O-3 position of the sugar is essential for hydrophobic interaction with PSA, LCH, and VFA. The substitution of methyl by nitrobenzyl leads to enhanced binding (1.7-16.7 times for the 2-O-substituted compounds and 7.9-40.5 times for the 3-O-substituted compounds) with the m-nitrobenzyl group contributing to maximum binding. A hydrophobic interaction is also involved between Con A and 2-O-nitrobenzyl derivatives, resulting in enhanced binding, but the corresponding 3-O-isomers bind poorly due probably to steric reasons. These results may be rationalized on the basis of the recently published X-ray data of Con A and VFA. The nitrobenzyl derivatives, after transformation to their azido analogs, have potential applications in the photoaffinity labeling of these lectins.

Chemical Phenomena↗

PubMed1992

Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium.

Antigens, Neoplasm↗

PubMed1991

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.

Amino Acid Sequence↗

PubMed1991

Interactions of five D-mannose-specific lectins with a series of synthetic branched trisaccharides.

The interaction of a series of synthetic, branched trisaccharides with five D-mannose-specific lectins was studied by precipitation-inhibition assay. The branched methyl alpha-D-mannotrioside, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man pOMe, the best inhibitor of the Con A-Dextran interaction, was 42 times more potent than alpha-D-ManpOMe, and 3-6 times more potent than the two trisaccharides substituted with D-glucosyl groups, and 8-15 times those with D-galactosyl groups. Surprisingly, methyl O-alpha-D-mannopyranosyl-(1----3)-alpha-D-mannopyranoside was bound to Con A 8-fold more avidly than methyl alpha-D-mannopyranoside. However, the related pea lectin (PSA) was singularly different from Con A in its carbohydrate-binding activity, showing no significantly enhanced binding to any of the sugars examined. The trisacchrides containing terminal, nonreducing, (1----3)-linked alpha-D-mannopyranosyl groups, i.e., alpha-D-Manp-(1----3)-[alpha-D-Glep-(1----6)]alpha-D-Manp OMe, alpha-D-Manp-(1----3)]-alpha-D-Galp-(1----6)]-alpha-D-ManpOMe++ +, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man pOMe, were the best inhibitors of the snowdrop lectin (GNA)-D-mannan precipitation system. On the other hand, all branched trisaccharides exhibited very similar inhibitory potencies toward the daffodil lectin (NPA)-D-mannan interaction, whereas alpha-D-Manp-(1----3)-[alpha-D-Galp-(1----6)]-alpha-D-ManpOMe++ + and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man pOMe were somewhat better inhibitors than the other branched trisaccharides of the amaryllis lectin (HHA)-D-mannan precipitation reaction. (ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗

PubMed1991

Use of the lectin from Amaranthus caudatus as a histochemical probe of proliferating colonic epithelial cells.

A newly isolated lectin, Amaranthus caudatus agglutinin (also called amaranthin or ACA), which binds to the Thomsen-Friedenreich antigen (T-antigen) and its sialylated variants, was used as a histochemical probe for proliferating cells in sections of human colonic tissues. Binding inhibition studies revealed that ACA binds to different sites on histological sections when compared to peanut agglutinin, which also recognizes the T-antigen. ACA bound selectively to the cells at the base of the colonic crypt [46 +/- 4% (SEM) of glands] which is the zone of proliferation in this tissue and preferentially labeled cytoplasmic and apical membrane glycoconjugates. Only 7 +/- 2% of the upper portions of the colonic crypts were labeled (P less than 0.001 compared to the base), and this was largely a result of extensive labeling in 2 of 23 samples studies. A marked increase in histochemical labeling by ACA was seen in adenomatous polyps and adenocarcinomas of the colon, in which 82 +/- 7 and 97 +/- 2% of the glandular units were labeled, respectively. Transitional mucosa and connective tissue adjacent to cancers were also labeled by ACA. Neuraminidase studies indicated that removal of sialic acid residues enhanced binding by peanut agglutinin, but not ACA, to glycoconjugates in cancer specimens. Specimens of colonic tissue from patients with familial adenomatous polyposis (FAP) were examined with ACA; 83 +/- 7% of adenomatous glands and 60 +/- 7% of glands in flat, normal-appearing tissue were labeled. Colonic tissues from persons at 50% risk for hereditary nonpolyposis colorectal cancer (HNPCC), FAP, and normal colons were studied and given "weighted average" labelling scores that ranged from 0-400 to accommodate variable intensity and distribution of labeling. Normal colons had a weighted average score of 65 +/- 33; FAP tissues had a score of 224 +/- 76 (P less than 0.001 compared to normal colon) and HNPCC tissues had a score of 74 +/- 70 (P less than 0.05 compared to normal colon). A group of five HNPCC cases had scores of 203 +/- 43 (P less than 0.001 compared to normal colon). ACA labels glycoconjugates in the proliferative region of normal human colonic epithelium and neoplastic lesions of the colon. The results of FAP and HNPCC tissues suggest that it may be useful for identifying foci of abnormal proliferation in familial colorectal cancer syndromes.

Adenocarcinoma↗

PubMed1991

Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins.

The sialic acid-specific leukoagglutinating lectin from the seeds of Maackia amurensis (MAL) has been studied by the techniques of quantitative precipitin formation, hapten inhibition of precipitation, hapten inhibition using an enzyme-linked immunosorbent assay, and lectin affinity chromatography. The ability of the immobilized lectin to fractionate oligosaccharides based on their content of sialic acid has also been investigated. Our results indicate that MAL reacts with greatest affinity with the trisaccharide sequence Neu5Ac/Gc alpha 2,3Gal beta 1,4GlcNAc/Glc. The lectin requires three intact sugar units for binding and does not interact when the beta 1,4-linkage is replaced by a beta 1,3-linkage nor when the "reducing sugar" of the trisaccharide is reduced. Results from enzyme-linked immunosorbent assays show that an N-acetyllactosamine repeating sequence is not required; however, the N-acetyllactosamine repeating sequence does appear to enhance the binding of MAL to a series of glycolipids. In addition, the sialic acid may be substituted with either N-acetyl or N-glycolyl groups without reduction in binding. The C-8 and C-9 hydroxyl groups of sialic acid do not play a role in binding as shown by the strong reaction of periodate-treated glycoproteins. Comparison of the specificity of the three sialic acid-binding lectins indicates that Limax flavus agglutinin binds to Neu5Ac in any linkage and in any position in a glycoconjugate, Sambucus nigra lectin requires a disaccharide of the structure Neu5Ac alpha 2,6Gal/GalNAc, and MAL has a binding site complimentary to the trisaccharide Neu5Ac alpha 2,3Gal beta 1,4GlcNAc/Glc, to which sialic acid contributes less to the total binding affinity than for either S. nigra lectin or L. flavus agglutinin.

Agglutination↗

PubMed1991

Differential expression of glycoproteins containing alpha-D-galactosyl groups on normal human breast epithelial cells and MCF-7 human breast carcinoma cells.

Cell surface glycoproteins were isolated from the lysates of 125I-labeled normal human mammary epithelial cells (NHMEC) and from the human breast carcinoma cell line MCF-7, of blood-group O phenotype, by affinity chromatography on Griffonia simplicifolia I lectin-Sepharose. Specific elution of glycoproteins from the column with methyl alpha-D-galactoside suggests the presence of alpha-D-galactosyl groups on these moieties. SDS-PAGE analysis of isolated glycoproteins revealed both quantitative and qualitative differences between glycoproteins from normal and malignant cells. Three major glycoproteins of Mr 180 kDa, 85 kDa and the 44 kDa were obtained from MCF-7 cells. The 180-kDa glycoprotein was absent in NHMEC and the 44-kDa glycoprotein was very weakly expressed in these cells. The only glycoprotein which was found in almost equal amount in the lysate from both normal and malignant cells was the 85-kDa glycoprotein. These results indicate differences between normal human mammary epithelial cells and one kind of malignant human mammary epithelial cells, in the expression of glycoproteins containing alpha-D-galactosyl groups, irrespective of blood-group phenotype; they also demonstrate that alpha-D-galactosyl group are expressed in a very restrictive manner on the surface of this tumor cell line.

Breast↗

PubMed1990

Physicochemical properties of amaranthin, the lectin from Amaranthus caudatus seeds.

Amaranthin is the lectin present in the seeds of Amaranthus caudatus, which specifically binds the T-disaccharide (Gal beta 1,3GalNAc alpha-O-). The lectin is composed of a single type of subunit with Mr = 33,000-36,000 (Rinderle et al., 1989). Equilibrium sedimentation (Mr = 62,900) and low-angle laser light scattering (Mr = 61,400) methods have been used to unambiguously establish the native multimeric structure of amaranthin as a homodimer. These absolute molecular weight methods and the calculated Stokes radius (27.2 A) indicate that the amaranthin dimer is highly compact relative to typical globular proteins, and thus, anomalous molecular weight values are obtained when simple size exclusion chromatography is used to determine the molecular weight of amaranthin. Studies with a homobifunctional cross-linking reagent and amaranthin further support the existence of a lectin homodimer. The stoichiometry of carbohydrate binding was determined to be one T-disaccharide-binding site per amaranthin subunit (Ka = 3.6 X 10(5) M-1). Amaranthin exhibits hydrophobic-binding properties as indicated by binding of 8-anilino-1-naphthalene-sulfonate (Ka = 3.6 X 10(3) M-1) and 6-toluidinyl-2-naphthalenesulfonate (Ka = 2 X 10(4) M-1). Serological studies suggest that amaranthin does not appear to be present in the stems or leaves of the A. caudatus plant, nor were there any indications for the presence of cross-reactive material.

Binding Sites↗

PubMed1990

Initiation of poly-N-acetyllactosamine chain biosynthesis occurs preferentially on complex multiantennary asparagine-linked oligosaccharides.

An N-acetyl-beta-D-glucosaminyltransferase activity involved in the initiation of poly-N-acetyllactosamine chain biosynthesis can be solubilized from Ehrlich ascites tumor cell microsomal membranes. The ability of this enzyme to act on linear and branched acceptor substrates has been studied. The results indicate that complex-type tri- and tetra-antennary oligosaccharides exhibiting the branching pattern beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)-[beta-D-Galp-(1----4)-beta- D- Glcp-NAc-(1----2)]-D-Man are the preferred substrates for the enzyme, and therefore, may represent the structures upon which the generation of poly-N-acetyllactosamine chains proceeds more efficiently.

Animals↗

PubMed1990

Crystallization and preliminary X-ray diffraction results of snowdrop (Galanthus nivalis) lectin.

Well-ordered single crystals have been grown for a mannose-specific lectin from snowdrop (Galanthus nivalis) bulbs in the presence of methyl-alpha-D-mannoside. The space group symmetry is consistent with the orthorhombic space group P2(1)2(1)2(1). The unit cell parameters are a = 140.0 A, b = 64.7 A, c = 62.0 A. The asymmetric unit can accommodate a tetramer. The functional molecule (50,000 daltons) consists of four identical subunits and is highly specific for alpha 1,3-linked mannose oligosaccharides.

Crystallography↗

PubMed1990

Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins.

The carbohydrate binding specificity of the daffodil (Narcissus pseudonarcissus; NPA) and amaryllis (Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with alpha-D-glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal alpha-D-galactosyl groups, a synthetic linear alpha-1,6-mannan, and an alpha-1,3-mannan (DP = 30). Treatment of the linear alpha-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal alpha-D-mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for alpha-D-mannosyl units whereas D-Glc and D-GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was alpha-1,6-linked mannotriose, which was twice as good an inhibitor as Man alpha 1,6Man alpha-O-Me and 10 times better than methyl alpha-D-mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than D-Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked alpha-mannosyl units. Various glycosylasparagine glycopeptides which contain alpha-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either alpha-1,3- or alpha-1,6-mannosyl residues were retarded on the immobilized HHA column; Man5-GlcNAc2-Asn [containing two Man alpha 1,3(Man alpha 1,6) units] bound to the HHA column. On the contrary, glycopeptides with hybrid type glycan chains were not retarded on either column. These two new lectins which differ in their fine sugar binding specificity from each other, and also from the snowdrop lectin, should prove to be useful probes for the detection and preliminary characterization of glycoconjugates on cell surfaces and in solution.

Binding, Competitive↗