Purification of a 24 kD parasitism-specific hemolymph protein from pharate pupae of the caribbean fruit fly,Anastrepha suspensa

We report purification of a 24 kD parasitism-specific protein (24 kD PSP) from pharate pupal hemolymph of the Caribbean fruit fly, Anastrepha suspensa, after parasitization by the braconid wasp, Diachasmimorpha (= Biosteres) longicaudata. We previously utilized isoelectric focusing (IEF) and two-dimensional (2-D) gel electrophoresis to demonstrate that the 24 kD PSP consists of two variants with pl 6.7 (more abundant) and pl 6.3. Purification of the more abundant 24 kD PSP variant was accomplished by Concanavalin A (Con A) sepharose B affinity chromatography followed by DEAE column chromatography. A second protocol, utilizing wheat germ agglutinin (WGA) sepharose 6MB affinity chromatography between the ConA and DEAE chromatographic steps, resulted in the purification of a partially deglycosylated form of the 24 kD PSP which retained its immunore-activity with anti-PSP serum but which exhibited a greater relative migration in sodium dodecyl sulfate (SDS)-PAGE than the pl 6.7 24 kD PSP variant. For structural studies both 24 kD PSP variants were purified from whole hemolymph by flat bed IEF followed by SDS-PAGE. Peptide cleavage profiles in 1-D SDS-PAGE after treatment with BNPS-skatole, CNBr, and endproteinases Lys-C and Asp-N were identical for both 24 kD PSP variants. Primary N-terminus sequences of at least the first 20 amino acid residues of both variants were identical. A secondary sequence of five amino acids residues was detected in both variants at Thr, the seventh amino acid residue from the N-terminus of the primary sequence. These data indicate that both 24 kD PSPs are glycoforms of a branched, apparently homogeneous polypeptide. © 1994 Wiley-Liss, Inc.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Lectin-Binding Sites Involved in Paramecium primaurelia Mating Pair Formation

ABSTRACT. Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating-competent mating type (mt) I and mt U cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific binding-monosaccharides, N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc), respectively, prevented the lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAc and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt I1 cells showed a high lectin-binding site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous lectin-binding site density spread on the cell surface, was also common to nonmating-competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating-competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt 11 cells.     

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.     

Lectin binding characterstics of mouse epididymal fluid and sperm extracts

Glycoproteins from luminal fluid of the mouse cauda epiciidymidis have been compared with glycoproteins from Triton X-100 extracts of mouse spermatozoa from varying regions of the epididymis, using lectins with specific affinity for different sugar residues. Concanavalin A recognizes 11 glycocomponents on Western blots of fractionated caudal fluid; wheat germ agglutinin (WGA) binds 12 proteins; Ulex europaeus agglutinin (UEA) binds seven; and Dolichos biflorus agglutinin (DBA) recognizes nine. Several of these glycoproteins display an affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues; whereas other proteins bind only one of the four lectins used. The results also show that some glycoproteins exhibit a higher affinity for particular lectins. Eight glycoproteins of similar mobility and lectin-binding characteristics are detected in Triton X-100 extracts of spermatozoa from different regions of the epididymis and in caudal fluid. The lectin affinity of some proteins appears or increases in spermatozoa from distal epididymal regions (54 kD, 32 kD), whereas the lectin affinity of others decreases (29 kD, 40 kD). There are differences in lectin affinities between proteins in sperm extracts and in caudal fluid. Some proteins show an affinity for three or four lectins in caudal fluid, but proteins of similar electrophoretic mobility in sperm extracts bind only one or two of the lectins. These data show that glycoproteins of similar mobility are present in caudal fluid and in Triton-X-100 sperm extracts, implying a potential interaction between caudal fluid components and epididymal sperm.     

亲和层析法分离纯化玉米精细胞质膜特异糖蛋白

 亲和层析法分离纯化玉米精细胞质膜特异糖蛋白陈丽邵邻相汪洁杨中汉（北京大学生命科学学院,北京１００８７１）ＩｓｏｌａｔｉｏｎａｎｄＰｕｒｉｆｉｃａｔｉｏｎｏｆＳｐｅｒｍＣｅｌＰｌａｓｍａＭｅｍｂｒａｎｅＧｌｙｃｏｐｒｏｔｅｉｎｓｏｆＺｅａｍａｙｓｂｙＡ...     

Hemocyte surface changes in the migratory grasshopper,Melanoplus sanguinipes in response to wounding and infection withBeauveria bassiana

Surface changes of hemocytes from the migratory grasshopper,Melanoplus sanguinipes (Fab.), following wounding and/or infection withBeauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA-binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3-fold in fungus-injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response ofM. sanguinipes to infection withB. bassiana are discussed.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Structural studies of the acidic oligosaccharides

Among the four acidic oligosaccharide fractions obtained by paper electrophoresis of the hydrazinolysate of the plasma membrane glycoproteins of rat erythrocytes, one was further separated into two by prolonged paper electrophoresis using 120-cm paper. Three fractions were mixtures of monosialyl oligosaccharides and two of disialyl oligosaccharides. After desialylation, their neutral portions were fractionated by Bio-Gel P-4 column chromatography and by affinity chromatography using a Con A-Sepharose column. Structural studies of the neutral oligosaccharides, thus obtained, indicated that at least 26 different complex-type oligosaccharides are present as a neutral portion of the acid oligosaccharides. Structurally they can be classified into bi-, tri-, and tetraantennary oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc_OT as their common cores. Galβ1 → 3Galβ1 → 4GlcNAc, Siaα2 → 3Galβ1 → 4GlcNAc, Siaα2 → 6Galβ1 → 4GlcNAc, and a series of Siaα2 → (Galβ1 → 4GlcNAcβ1 → 3)_n · Galβ1 → 4GlcNAc were found as their outer chains. Their structures together with the structures of neutral oligosaccharides reported in the preceding paper indicated that the outer chain moieties of the asparagine-linked sugar chains of rat erythrocyte membrane glycoproteins are formed not by random concerted action of glycosyl transferases in Golgi membrane but by the mechanism in which the formation of one outer chain will regulate the elongation of others.     

Biosynthesis of the cancer-associated sialyl-Lea antigen

A cancer-associated glycolipid antigen defined by monoclonal antibody 19-9 has the structure NeuAc alpha 2-3Gal Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer. We have (formula; see text) studied its biosynthesis by testing the capacity of a crude microsomal fraction of SW 1116 cells to catalyze the addition of fucosyl or sialyl residues from GDP-fucose or CMP-sialic acid to glycolipid or oligosaccharide precursors. When the tetrasaccharide NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LSTa) is incubated with GDP-[14C]fucose and SW 1116 microsomes, a 14C-labeled oligosaccharide is formed that can be separated from the incubation mixture on an affinity column containing antibody 19-9 bound to protein A-Sepharose. The product migrates slower than LSTa when analyzed by paper or thin-layer chromatography. After treatment with neuraminidase, it co-migrates with the pentasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (formula; see text) (LNF II) in both chromatographic systems. Similar experiments demonstrate that SW 1116 microsomes catalyze the addition of a sialyl residue to the tetrasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc to form LSTa. However, when LNF II is incubated with CMP-[14C]sialic acid and SW 1116 microsomes, no 19-9-active product is detected by affinity chromatography or by paper or thin-layer chromatography. Results using glycolipid precursors are consistent with these findings and also demonstrate the presence of the Lewis fucosyltransferase in SW 1116 cells. Thus, the biosynthesis of the sialyl-Lea antigen proceeds by addition of sialic acid to a type 1 precursor chain by a sialyltransferase, followed by addition of fucose by the Lewis fucosyltransferase.     

Cell surface saccharide differences in drug-susceptible and drug-resistant strains of Trichomonas vaginalis.

Surface carbohydrates of drug-resistant and drug-susceptible strains of Trichomonas vaginalis were analysed using lectins. The presence of D-GalNAc, D-Gal and mannose-like residues was detected in T. vaginalis. Marked differences in exposed surface carbohydrates were documented, e.g. wheat germ agglutinin (WGA) selectively agglutinated the drug-susceptible strain whereas drug-resistant parasites reacted preferentially with concanavalin A (Con A). In drug-resistant, but not in drug-susceptible strains, trypsinization induced the appearance of soybean agglutinin. Binding studies using fluorescein-labelled WGA and Con A essentially confirmed the agglutination experiments. Both the intense cell agglutination and the fluorescent WGA-binding displayed by a drug-susceptible strain, were completely nullified by neuraminidase treatment, suggesting the presence of an exposed sialic acid moiety on the T. vaginalis surface.     

Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins.

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Phar) phenotype is stable in the absence of selection; its frequency in a sensitive-population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal alpha-mannose residues is dramatically reduced (less than or equal to 5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytoxocity of concanavalin A (Con A); (d) an increased ability to bind 125I-Con A; and (e) decreased surface galactose residues - all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistance closes from each of two complementary CHO auxitrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC, or LCA, and 4-5 "lectin-resistance" phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.     

A novel sialylhexasaccharide from human milk: purification by affinity chromatography on immobilized wheat germ agglutinin

A sialylhexasaccharide fraction (S-5) of human milk was obtained as described by A. Kobata and V. Ginsburg [(1972) Arch. Biochem. Biophys. 150, 273-281] and labeled by reduction with NaB[3H]4. When subjected to affinity chromatography on immobilized wheat germ agglutinin (WGA), a single component representing 60% of the S-5 fraction was retarded by the column. The asialo derivative of the WGA-retarded oligosaccharide had a higher affinity for the WGA column than the native sialyloligosaccharide. The neutral hexaose was identified as lacto-N-neohexaose by sequential exoglycosidase digestions in combination with gel filtration analyses of digestion products. Enzymatic removal of the nonsialylated branch of the intact sialyloligosaccharide by jack bean beta-galactosidase and beta-N-acetylhexosaminidase resulted in a single sialyl[3H]tetraose which was identified as sialyltetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcO[3H]) by cochromatography with authentic standard and specific antibody binding. Independent evidence for the structure of the sialylhexasaccharide was obtained by 500-MHz1H NMR spectroscopy of the WGA-purified oligosaccharide before and after neuraminidase digestion. The structural data are consistent with the following, previously undescribed, sialylhexaose in human milk: (formula; see text).     

Detection of sugar residues in lizard tooth germs (Liolaemus gravenhorsti) using lectin histochemistry

The appearance, cellular distribution, and changes of sugar residues during tooth development in adults of the polyphyodont, Liolaemus gravenhorsti, were investigated by using horseradish–peroxidase–conjugate lectins (lectin–HRP). With Con A (Canavalia ensiformis), the ameloblasts (late bell stage) show granular supranuclear positivity and also at the Golgi zone and on their tomes process. Reactivity also appears at the apical surface of the odontoblasts and odontoblastic process. With WGA (Triticum vulgaris), the tooth germs (late bell stage) show cytoplasmatic granular positivity in the ameloblast cells, Golgi regions, and in a lesser extent of the cytoplasm. Also, the apical surface and the odontoblastic process react. WGA reaction is depressed following sialidase treatment. The significance in tooth germs of α-D -mannose, α-D -glucose as well as β-D -N-acetylglucosamine and sialic acid is difficult to ascertain. These oligosaccharides may have some significance in odontogenesis. In fact, Con A-HRP- and WGA-HRP-binding components in ameloblasts and odontoblasts may be functionally related to molecules that are thought to contribute to odontogenesis in lizards. © 1994 Wiley-Liss, Inc.     

Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF

The serum of the freshwater prawn contains a sialic acid specific lectin (MrL) that agglutinates erythrocytes from rat and rabbit, as well as some Gram negative and positive bacterial strains. In this work, we performed the chemical characterization of the MrL purified by affinity chromatography on stroma from rat erythrocytes and by ion exchange chromatography. In its active form, MRL is a dimeric glycoprotein with 9.5 kDa per subunit. The amino acid sequence of the lectin was deduced from peptides obtained after trypsin treatment by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF). The predicted amino acid sequence of the lectin showed 54% homology with the hyperglycemic hormone from Macrobrachium rosenbergii. It also showed homology with the variable region of the human immunoglobulin kappa (22%) and lambda (27%) light chains. The lectin is a glycoprotein with 11% (w/w) carbohydrate content and is constituted by Gal, Man, GlcNAc, GalNAc and NeuAc in a molar ratio of 4:3:2:1:0.6. The primary structure of the carbohydrate chains of the lectin from the freshwater prawn was determined by affinity chromatography of MrL-glycopeptides on Con A and LCA lectin columns, which indicated that the main carbohydrate chains conforming the lectin are N-glycosidically linked. Man3 GlcNAc2.1 oligosaccharides were the most abundant structures with 57%) followed by Gal1.3 Man3 GlcNAc2.8 with 24%. Our results suggest that the freshwater prawn possess a lectin in the hemolymph plasma, related to those from the immunoglobulin superfamily.     

Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells

Summary Expression of various sugar residues on the plasma membrane of frog (Rana perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectins. In paraffin-embedded tissue, mannose residues (stained by Concanavalin A) were detected at the keratinocyte cell surface in all epidermal strata. However,Lens culinaris agglutinin (LCA), also specific for mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), labelled with wheat germ agglutinin (WGA), was maximum at the cell surface of basal cells and progressively decreased through the stratum spinosum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, labelled withGriffonia simplicifolia I (GS I) andGlycine max (SBA) agglutinins, respectively, were expressed according to the degree of differentiation in amphibian epidermal cells. Sialic acid-containing glycoproteins, labelled withLimax flavus agglutinin (LFA), were found in the outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding patterns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane glycoproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and glycoproteins containing terminal Gal residues, that are expressed according to the degree of differentiation in frog epidermis, have been partially purified by affinity chromatography on a GS I-Sepharose 4 B column. The purified fraction was composed by four acidic glycoproteins with isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five major protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to the a and subunits of amphibian epidermal Na⁺,K⁺-ATPase as demonstrated by specific staining with a polyclonal antibody against the catalytic subunit of pig kidney proton pump and staining with lectins GS I, GS II, and WGA. Possible relationships between higher molecular weight proteins and the constituents of intramembranous particles from the outermost plasma membranes of the replacement cell layer and the stratum corneum are also discussed.Abbreviations BSA bovine serum albumin - CHAPS (3-[(cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate) - Con A Canavalia ensiformis agglutinin - DTT dithiothreitol - Gal galactose - GalNAc N-acetyl-D-galactosamine - GlcNAc N-acetyl-D-glucosamine - GS I Griffonia simplicifolia agglutinin I - GS II Griffonia simplicifolia agglutinin II - HRP horseradish peroxidase - LFA Limax flavus agglutinin - LCA Lens culinaris agglutinin - NDPAGIF non-denaturing polyacrylamide gel isoelectric focusing - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase - PBS phosphate buffered saline - PMSF phenyl methyl sulphonyl fluoride - RCL replacement cell layer - SBA soybean agglutinin (Glycine max) - SB stratum basal - SDS sodium dodecyl sulphate - SG stratum granulosum - SS stratum spinosum - UEA I Ulex europaeus agglutinin I - WGA wheat germ (Triticum vulgaris) agglutinin     

Cell Surface Saccharides in Three Phytomonas Species Differing in Host Specificity

ABSTRACT. Cell surface carbohydrates of three phytoflagellates, Phytomonas francai. Phytomonas serpens and Phytomonas sp. from different hosts including cassava, coreid insect Phthia picta and the milkweed plant Euphorbia hyssopifolia, respectively, were analysed by agglutination assays employing a battery of highly purified lectins with affinity for receptor molecules containing N-acetylglucosamine (d-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose, mannose-like (D-Man-like) residues and fucose, and by binding assay using radiolabeled [¹²⁵I]-wheat germ agglutinin (WGA) and fluorescent WGA lectin, as well as glycosidases of known sugar specificity, Escherichia coli K with mannose-affinity fimbrial lectin was also used as an agglutination probe. In general, the presence of D-GlcNAc. D-GalNAc and D-Man-like residues was detected in the phytomonads' plasma membrane. These sugar moieties were confirmed in whole cell hydrolysates as assessed by gas-liquid chromatography (GLC) which in addition, also showed the presence of galactose and xylose. However, marked differences in cell surface carbohydrate structures were observed. Wheat germ agglutinin, which binds to sialic acid and/or d-GlcNAc-containing residues, shows selective agglutinin activities for P. francai and Phytomonas sp., while Bandeiraea simplicifolia II agglutinin (which recognizes d-GlcNAc units) specifically bound to Phytomonas sp. Helix pomatia agglutinin which binds to D-GalNAc-containing residues reacted preferentially with Phytomonas sp. and P. serpens. Con A, which recognizes D-Man-like receptors, agglutinates all the phytomonads; however, the higher interaction was observed with Phytomonas sp. P. francai was selectively agglutinated in the presence of E. coli fimbrial lectin. Fluorescence WGA binding was significantly decreased by N-acetylglucosaminidase activities and the cell agglutination was not altered by neuraminidase treatment, suggesting the presence of an exposed D-GlcNAc moiety on the P. francai and Phytomonas sp. surfaces. Binding studies with [¹²⁵I]-WGA essentially confirmed the fluorescence WGA binding and agglutination assays.     

Interactions of wheat-germ agglutinin with GlcNAc beta 1,6Gal sequence

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.     

Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of lectins

Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha^R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent Pha^R clones. These clones also exhibit (a) a decreased ability to bind [¹²⁵I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind¹²⁵I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A^R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Application of a lectin from the mushroom Polysporus squamosus for the histochemical detection of the NeuAcα2,6Galβ1,4Glc/GlcNAc sequence of N-linked oligosaccharides: a comparison with the Sambucus nigra lectin

The lectin from the mushroom Polysporus squamosus (PSL) has an extended carbohydrate combining site, which exhibits a high specificity and affinity toward the NeuAc5alpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequence of asparagine-linked oligosaccharides. Therefore, PSL should be a superior reagent to the lectin from Sambucus nigra (SNA), which does not discriminate between alpha2,6-linked NeuAc5 present either in asparagine- or serine/threonine-linked oligosaccharides. We have prepared a digoxigenin-conjugated PSL and applied it for histochemistry and blotting. We observed a more restricted staining pattern by PSL as compared to SNA in paraffin sections from different rat organs. Pretreatment of sections with N-glycanase F abolished PSL staining indicating that it interacts only with asparagine-linked oligosaccharides. Furthermore, PSL staining was neuraminidase sensitive. In contrast, SNA staining was only partially sensitive to N-glycanase F pretreatment demonstrating that it was in part due to alpha2,6-linked NeuAc5 present in serine/threonine-linked oligosaccharides. The most striking observation in this regard was that PSL, in contrast to SNA, did not stain the mucus of sheep submandibular gland, which is extremely rich in serine/threonine-linked Neu5Acalpha2,6N-acetylgalactosamine. Furthermore, in some tissues neuraminidase pretreatment resulted in increased intensity of SNA staining probably due to binding to exposed terminal N-acetylgalactosamine residues. Collectively, these results indicate that PSL is a useful tool for the histochemical detection of alpha2,6-linked NeuAc5 in asparagine-linked oligosaccharides.     

Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB₅ toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.