Oligosaccharide Specificity Research Articles

1206 The role of neurotrophin for the neurite extension and cell migration of cerebellar granule cells in vitro

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Affinity electrophoretic determination of oligosaccharide specificity of Butea monosperma agglutinin.

The oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two-dimensional lectin affinity electrophoresis of alpha-fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex-type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man alpha1->3 arm, monosialyl residue of the Man alpha1->6 arm, monosialyl residue of the Man alpha 1->3 arm and bisecting G1cNAc, and disialyl residues. BMA did not recognize Neu5Ac alpha2->6 and Neu5Ac alpha2->3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy-associated alteration of AFP sugar chains.

Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.

The soluble, calcium-binding protein calreticulin shares high sequence homology with calnexin, a transmembrane chaperone of glycoprotein folding. Our experiments demonstrated that calreticulin, like calnexin, associated transiently with numerous newly synthesized proteins in the endoplasmic reticulum. The population of proteins that bound to calreticulin was partially overlapping with those that bound to calnexin. Hemagglutinin (HA) of influenza virus was shown to associate with both calreticulin and calnexin. Using HA as a model substrate, it was found that both calreticulin- and calnexin-bound HA corresponded primarily to incompletely disulfide-bonded folding intermediates and conformationally trapped forms. Binding of all substrates was oligosaccharide-dependent and required the trimming of glucose residues from asparagine-linked core glycans by glucosidases I and II. In vitro, alpha-mannosidase digestion of calreticulin-bound HA indicated that calreticulin was specific for monoglucosylated glycans. Thus, calreticulin appeared to be a lectin with similar oligosaccharide specificity as its membrane-bound homologue, calnexin. Both are therefore likely to play an important role in glycoprotein maturation and quality control in the endoplasmic reticulum.

Solubilization of Functional Plasma Membrane-Localized Hepta-[beta]-Glucoside Elicitor-Binding Proteins from Soybean

Total membranes prepared from roots of soybean (Glycine max L.) seedlings have previously been shown to contain proteinaceous binding site(s) for a hepta-beta-glucoside elicitor of phytoalexin accumulation. The hepta-beta-glucoside elicitor-binding proteins have now been shown to co-migrate with a plasma membrane marker enzyme (vanadate-sensitive H(+)-ATPase) on linear sucrose density gradients. With the use of detergents, the elicitor-binding proteins have been solubilized in functional form from soybean root membranes. The nonionic detergents n-dodecylsucrose, n-dodecylmaltoside, and Triton X-114, at concentrations of 5 to 10 mg/mL, each solubilizes between 50 and 60% of the elicitor-binding activity in a single extraction of the membranes. A zwitterionic detergent, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (ZW 3-12), also solubilizes about 40% of the total binding activity at detergent concentrations between 1 and 2 mg/mL, but the total binding activity recovered is only approximately 50% of that recovered with the nonionic detergents. The elicitor-binding proteins solubilized with either n-dodecylsucrose or ZW 3-12 retain the high affinity for radiolabeled hepta-beta-glucoside elicitor (apparent dissociation constant [Kd] = 1.8 nM and 1.4 nM, respectively) that was observed with the membrane-localized binding proteins (apparent Kd = 1 nM). Competitive ligand-binding experiments with several structurally related synthetic oligoglucosides demonstrate that the solubilized binding proteins retain specificity for elicitor-active oligosaccharides, irrespective of the detergent used for solubilization. Moreover, the binding affinities of the oligoglucosides for the solubilized binding proteins correlate well with their abilities to induce phytoalexin accumulation in soybean cotyledon tissue. Gel-permeation chromatography of n-dodecylsucrose-solubilized elicitor-binding proteins demonstrate that the bulk of the elicitor-binding activity is associated with large detergent-protein micelles (relative molecular weight > 400,000). Our results suggest that n-dodecylsucrose is a suitable detergent for solubilizing elicitor-binding proteins from soybean root membranes with minimal losses of binding activity. More importantly, we demonstrate that solubilization does not significantly after the binding properties of the proteins for elicitor-active oligoglucosides.

Substrate Specificity of the Heparin Lyases from Flavobacterium heparinum

A detailed knowledge about the substrate specificities of the heparin lyases is necessary when using these enzymes as tools for elucidating the sequence of heparin and heparan sulfate. The substrate specificity of heparin lyases I, II, and III have been profiled with structurally defined, heparin-derived oligosaccharides. The primary substrate specificities of heparin lyases I and III require the presence of 2-O-sulfated α-L-idopyranosyluronic acid and β-D- glucopyranosyluronic acid residues, respectively, at the linkages being cleaved. Heparin lyase II demonstrates an intriguingly broad primary specificity for oligosaccharides, acting at linkages containing α-L-idopyranosyluronic and β-D-glucopyranosyluronic acid as well as at linkages containing α-L-galactopyranosyluronic acid residues. In addition to their primary specificities, each lyase also demonstrates secondary specificities under forcing conditions. Differences in the sulfation pattern within uronic acid residues and sulfation of adjacent residues has profound impact on the ease of lyase cleavage of a glycosidic linkage. Specifically, heparin lyases I and III exhibit secondary specificity for oligosaccharides containing an unsulfated α-L-idopyranosyluronic acid residue. The lack of sulfation on residues adjacent to the linkage undergoing cleavage increases the action of heparin lyase III on a glycosidic linkage. In contrast, reduced sulfation on adjacent residues make glycosidic linkage resistant to heparin lyase I. The primary and secondary specificity can be rationalized on the basis of most favorable solution conformation of the uronic acid residues.

Oligosaccharide specificity of normal human hepatocyte α1–3 fucosyltransferase

A purified α1–3 fucosyltransferase (α1–3 FT) was recovered in the Golgi fraction of isolated hepatocytes from normal human liver tissue. The efficiency of purification was controled by measurement of fucose transfer to asialotransferrin (for α1–3 FT), to phenyl-β- d-galactose (for α1–2 FT) and to 2′ fucosyl lactose (for α1–3/4 FT). The initial hepatocyte isolation step got rid of 97% and 94% of α1–2 and α1–3/4 total liver FT, respectively. After Golgi enrichment (26-fold purification and a yield of 7.6%), α1–3 FT extract expressed a specific activity of 2 pM/min per mg protein. When incubated in optimized conditions with type 1, 2 or 6 oligosaccharide acceptors (10 mM), hepatocellular α1–3 FT efficiently transferred fucose to N-acetyllactosamine and its 3′sialylated derivative, but poorly to lactose. When incubated with neutral or sialylated biantennary N-glycans, the enzyme expressed the highest affinity (K m = 0.38 mM) for the 3′bisialylated derivative. This suggests that hepatocellular α1–3 FT is involved in the synthesis of sialosyl Le x determinants on cirrhotic α1AGP.

Analysis of sequence variation among legume lectins: A ring of hypervariable residues forms the perimeter of the carbohydrate-binding site

Twelve plant lectins from the Papilionoideae subfamily were selected to represent a range of carbohydrate specificities, and their sequences were aligned. Two variability indices were applied to the aligned sequences and the results were analysed using the three-dimensional structures of concanavalin A and the pea lectin. The areas of greatest variability were located in the carbohydrate-binding site region, forming a perimeter around a well-conserved core. These residues are inferred to be specificity determining, in the manner of antibodies, and the most variable position corresponded to Tyr100 in concanavalin A, a known ligand contact residue. In addition to the five peptide loops known to form the binding site from crystallographic studies, a sixth segment with variable residues was located in the binding-site region, and this may contribute to oligosaccharide specificity. In their overall composition, the lectin sites resemble those of the sugar-transport proteins rather than antibodies. The prospects for modelling lectin binding sites by the methods used for antibodies were also assessed.

Localization and characterization of the carbohydrate-binding site of the porcine lymphocyte mannan-binding protein

Mannan-binding proteins found in the liver and serum of several vertebrate species are supposed to play an important role in the intracellular transport of glycoproteins, as well as in several protective reactions including complement activation and elimination of various pathogens. To study these protective functions at molecular level it is necessary to understand the fine oligosaccharide specificity and mutual relation among various forms of these soluble lectins. We have isolated mannan-binding protein as peripheral membrane proteins of porcine lymphocytes. This lectin was purified to homogeneity and shown to possess many properties in common with the well studied rat liver proteins (mol. mass, subunit composition and general organization of the molecule). Binding studies performed with three series of defined oligosaccharides (high mannose, hybrid type, and complex) on native lectin molecules as well as isolated carbohydrate-binding domains revealed distinctive features of this mannan-binding protein, including its impaired ability to bind the oligosaccharide ligand after reduction and decyclization at core N-acetyl- d-glucosamine 1.

Oligosaccharide-binding molecules on the surface of human hemopoietic and lymphoid cells

High oligosaccharide specificity for binding carbohydrate probes (biotinylated polyacrylamide with carbohydrates attached) with human hemopoietic and lymphoid cells is shown. Of 15 probes studied those bearing blood group trisaccharides, A and B, bound most intensely. In addition, transformed (leukemic and lymphoid) cells interacted more strongly than normal ones.

Interaction of lectins withYersinia pestis strains

The ability of lectins to interact with Yersinia pestis strains isolated from rodent fleas and human biological fluids, obtained from different geographic areas, was examined. Lectins of Canavalia ensiformis, Ulex europaeus, Phaseolus vulgaris, and Triticum vulgaris, as well as a new autochthonous lectin of Swartzia pickellii of undefined specificity, were used. Most of the Y. pestis strains did not agglutinate with U. europaeus or C. ensiformis lectin. However, P. vulgaris lectin agglutinated suspensions of all the bacillus strains used. Fifteen of the 19 strains tested positive for assays using S. pickellii lectin. It is believed this is the first report of Y. pestis strain agglutination by lectins. A similar agglutination pattern was obtained for lectins with specificity for oligosaccharides containing N-acetylglucosamine and S. pickellii lectin, which did bind to the affinity matrix chitin, a polysaccharide of N-acetylglucosamine. The use of bacterial strains and commercial lectins of defined specificity may be an approach to providing evidence about the lectin binding sites of undefined monosaccharide specificity.

Two‐dimensional lectin affinity electrophoresis of α‐fetoprotein: Characterization of erythroagglutinating phytohemagglutinin‐dependent microheterogeneity forms

By means of two-dimensional lectin affinity electrophoresis of human alpha-fetoprotein (AFP) from different sources, AFP bands separated with erythroagglutinating phytohemagglutinin (E-PHA) were further characterized with other lectins of known oligosaccharide specificities. The results with a cord serum AFP revealed that not only AFP-P2 (E-PHA-nonreactive) but also AFP-P4 and P5 (E-PHA-reactive) had affinities for Concanavalin A (Con A) and Allomyrina dichotoma lectin (allo A), indicating that the cord serum AFP has nonbisected biantennary complex-type oligosaccharides with the terminal galactose on Man alpha 1----6 residue sialylated at the C-6, but not C-3, position. On the other hand, the results with a hepatoblastoma (HUH-6 C1-5 cell line) AFP showed that not only AFP-P5 but also AFP-P1 (E-PHA-nonreactive) and P3 (E-PHA-less reactive) had Con A-nonreactive AFP and that AFP-P1 had AFP-A1 (allo A-nonreactive) and AFP-A2 (allo A-less reactive), and AFP-P3 and P4 had AFP-A1s (allo A-nonreactive), as main components, in addition to the spots of cord serum AFP. Most of the E-PHA-dependent bands of AFP were further subdivided with Lens culinaris agglutinin (LCA-A) into LCA-A-reactive, weakly reactive and nonreactive spots. Similar results were obtained with AFP preparations from hepatocellular carcinomas and other malignancies, indicating that the bisected bi-(or tri- and tetra-) antennary sugar chains with the exposed terminal galactose of the Man alpha 1----6 arm as well as those with the C-3 sialylated galactose residues could be expressed in AFP upon malignant transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lectinohistochemistry of human bladder cancer: loss of lectin binding structures in invasive carcinomas.

With the purpose of studying changes in the expression of glycoconjugate structures in urothelium, nine different lectins (PNA, WGA, VFA, GSA II, STA, UEA I, LCA, DBA and HPA) with specificity for mono- or oligo-saccharides were used on formalin-fixed, paraffin-embedded tissue sections from 47 patients who had undergone surgical resection for bladder tumors and on normal urothelial biopsies from 10 patients. The tumors were graded and a lectinohistochemical method using biotinylated lectins and avidin-biotin-peroxidase complex was used to demonstrate the lectin binding. Positive staining reactions of cells in cytoplasm and on membranes were evaluated in the basal, the intermediate, and the luminal cell layers, respectively. In both normal and atypical urothelium lectin binding predominated in the luminal cell layer and decreased towards the basal cell layer. In normal urothelium all lectins stained greater than 66% of the cells in the luminal cell layer in cytoplasm and between 5 and 100% of the cells on membranes depending on the lectin used. A gradual loss of lectin-binding structures was seen with increasing grade of atypia. The range of this decrease varied considerably from one lectin to another, but it was consistently found that the percentage of cells stained in cytoplasm and on membranes decreased. A significantly lower percentage of cells stained in cytoplasm was found in invasive tumor cell-islands compared to normal urothelium. In invasive tumor cell-islands staining of cells on membranes was completely absent, except for HPA lectin that stained less than 10% of the cells. In conclusion, we demonstrate a dramatic decrease in lectin-binding carbohydrate structures associated with urothelial malignant progression.

Host cell-induced differences in O-glycosylation of herpes simplex virus gC-1 : I. Structures of Nonsialylated HPA- and PNA-Binding Carbohydrates

Lectins with narrow oligosaccharide specificities were established as probes to study the host cell influence on the biosynthesis of O-linked oligosaccharides of the herpes simplex virus type 1 (HSV-1)-specified glycoprotein C (gC-1). We found that only gC-1 and no other glycoprotein bound to the peanut lectin (PNA), with main specificity for Gal(β1–3)GaINAc. Previously, we have shown that only gC-1 binds to the Helix pomatia lectin (HPA), with main specificity for terminal GaINAc. The O-linked oligosaccharides binding to PNA and HPA were released by alkaline borohydride treatment and characterized. A structural determination of these oligosaccharides showed that the HPA-binding carbohydrates were monosaccharides (GaINAc), and that the PNA-binding oligosaccharides were disaccharides with the structure Gal-GaINAc. The PNA- and HPA-binding oligosaccharides were arranged as Pronase-resistant clusters on gC-1, consisting of about seven individual, adjacent oligosaccharides. In addition to these disaccharides, Pronase-resistant PNA-binding glycopeptides of gC-1 also contained neutral trisaccharides. Larger O-linked oligosaccharides, binding to the wheat germ lectin, were found in gC-1, but not in proximity to the PNA-binding ones. It was concluded that the lectins mentioned should be useful probes in screening HSV-infected cells of different lineages for differences in processing of 0-linked oligosaccharides.

Lectin affinity high-performance liquid chromatography: Interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH 4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II.

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.

Detection and quantification of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidases

A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CHO)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4'-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1-2 nmol) of detection of peptide-N4-(N-acetylglucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the corresponding enzyme from almonds.

Human influenza A virus hemagglutinin distinguishes sialyloligosaccharides in membrane-associated gangliosides as its receptor which mediates the adsorption and fusion processes of virus infection. Specificity for oligosaccharides and sialic acids and the sequence to which sialic acid is attached.

Human influenza A virus isolates bearing antigenically different H1 (A/PR/8/34), H2 (A/Japan/305/57), and H3 (A/Aichi/2/68, A/X-31) hemagglutinin serotypes caused extensive hemagglutination, low pH fusion, and hemolysis of asialoerythrocytes reconstituted with gangliosides. Sialylparaglobosides (IV3NeuAc-nLc4Cer, IV6NeuAc-nLc4Cer), I-active and i-active (VI3NeuAc-nLc6Cer) gangliosides, and GM3-NeuAc commonly exhibited significant specific receptor activity toward the viruses. A/PR/8/34 recognized IV3NeuAc-nLc4Cer containing the NeuAc alpha 2-3Gal sequence preferentially over IV6NeuAc-nLc4Cer containing NeuAc alpha 2-6Gal, whereas the other two recognized the NeuAc alpha 2-6Gal sequence preferentially over NeuAc alpha 2-3Gal. Responsiveness of erythrocytes labeled with gangliosides containing NeuGc to the viruses used was considerably lower than that of erythrocytes labeled with gangliosides containing NeuAc. The activities of GM1a, GM2, and GD1b bearing NeuAc on inner galactose of the ganglio series core were also very low. These results indicate that sialyloligosaccharides of IV3NeuAc-nLc4Cer, IV6NeuAc-nLc4Cer, I-active ganglioside, and VI3NeuAc-nLc6Cer in addition to GM3-NeuAc and GM1b-NeuAc (Suzuki, Y., Matsunaga, M., and Matsumoto, M. (1985), J. Biol. Chem. 260, 1362-1365; Suzuki, Y., Matsunaga, M., Nagao, Y., Taki, T., Hirabayashi, Y., and Matsumoto, M. (1985) Vaccine 3, 201-203) are functional receptor determinants toward hemagglutinin of human influenza A viruses, and the viruses differentiate microdomains of the gangliosides, such as the sialic acid species (NeuAc, NeuGc) and the sequence of sialic acid linkages (NeuAc alpha 2-3Gal, NeuAc alpha 2-6Gal).

Structural requirements for sulfation of asparagine-linked oligosaccharides of lutropin.

Human and bovine pituitary glycoprotein hormones (lutropin, follitropin, and thyrotropin) contain varying amounts of N-acetylgalactosamine and sulfate. The sulfate on asparagine-linked oligosaccharides of bovine lutropin (bLH) is present exclusively on GalNAc in the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha. We have examined the structural requirements for sulfation of bLH oligosaccharides by using a reconstituted cell-free system. After cleavage from the protein, oligosaccharides containing the sequence GalNAc(beta 1-4)Glc-NAc(beta 1-2)Man alpha were sulfated by enzymes in pituitary membranes. Addition of one or two sulfates was observed, depending upon the number of GalNAc acceptor sites on the oligosaccharide. Neither GalNAc alone nor oligosaccharides devoid of GalNAc were sulfated. Membranes from placenta or liver did not sulfate oligosaccharides released from bLH, indicating that the sulfating activity is pituitary-specific. The lack of peptide dependence for sulfation, in conjunction with the oligosaccharide specificity, suggests that the sequence GalNAc(beta 1-4)GlcNAc(beta 1-2)Man alpha contains the recognition signal for the sulfotransferase(s).

Characterization of microsomal and cytosolic α-1,2-mannosidases from mung bean hypocotyls

Microsomal and cytosolic α-mannosidase activities, which hydrolyze α-1,2-mannosylmannose linkages in the Man 5GlcNAc 2 oligosaccharide, have been isolated from homogenates of mung bean hypocotyls. The α-1,2-mannosidase activities were readily distinguished from previously described aryl α-mannosidases by several criteria. They were optimally active in the presence of Ca 2+ between pH 5.5 and 6, they were inhibited by Zn 2+, and they had essentially no activity with p-nitrophenyl-α-mannoside. The microsomal and cytosolic α-1,2-mannosidases demonstrated specificity for oligosaccharides with terminal nonreducing α-1,2-mannosyl linkages, and they were inhibited by mannosyl-mannose disaccharides, with the inhibition decreasing in the order of α-1,2- > α-1,3- > α-1,6-mannosyl-mannose. The cytosolic α-1,2-mannosidase activity, which was present in the 100,0000 g supernatant, was separated from the aryl α-mannosidase by ammonium sulfate precipitation. The microsomal α-1,2-mannosidase, which was tightly associated with the particulate fraction, was solubilized with Triton X-100 and 0.2 m KCl. The two α-1,2-mannosidase activities were readily differentiated by gel-filtration chromatography. The solubilized microsomal enzyme chromatographed in approximately the same position as a M r 460,000 globular protein whereas the cytosolic enzyme was eluted in a retarded position, indicating a much smaller protein.

Characterization of a mannose and N-acetylglucosamine-specific lectin present in rat hepatocytes.

We have isolated and characterized a Man/GlcNAc-specific lectin from rat liver. The specificity of this lectin resembles that of the previously reported Man/GlcNAc-specific receptor activity present on the surface of hepatic reticuloendothelial cells when tested with simple sugars, but can be distinguished from the latter by utilizing characterized glycopeptides and glycoproteins. The lectin isolated from liver originates predominantly from hepatocytes rather than reticuloendothelial cells. It is found in microsomes upon subcellular fractionation and is not expressed on the surface of isolated hepatocytes. In addition, specific antisera to the lectin do not prevent endocytosis by hepatic reticuloendothelial cells. The lectin can be solubilized by treatment of whole liver or isolated hepatocyte homogenates with buffers containing 0.2 M NaCl in the absence of any detergent, indicating that it may be either a soluble protein or a peripheral membrane protein. Based on the physical properties, oligosaccharide specificity, and subcellular location, this lectin does not appear to be the same as the Man/GlcNAc-specific activity which mediates endocytosis by hepatic reticuloendothelial cells and presumably has a different function.