Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thin-layer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal alpha-linked GalNAc residues did not bind to globoside (terminal beta 1-3GalNAc) but did bind the ganglioside GM2 and its asialo derivative which have terminal beta 1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM2. The interactions of these lectins with the glycolipid-derived, 3H-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I-Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A1 and A2 cells.     

Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis

The presence of capsular exopolysaccharide (EPS) in Mollicutes has been inferred from electron micrographs for over 50 years without conclusive data to support the production of complex carbohydrates by the organism. Mycoplasma pulmonis binds the lectin Griffonia simplicifolia I (GS-I), which is specific for terminal β-linked galactose residues. Mutants that failed to produce the EPS bound by GS-I were isolated from a transposon library. All of the mutants had the transposon located in open reading frame MYPU_7410 or MYPU_7420. These overlapping genes are predicted to code for a heterodimeric pair of ABC transporter permeases and may code for part of a new pathway for synthesis of EPS. Analysis by lectin-affinity chromatography in conjunction with gas chromatography demonstrated that the wild-type mycoplasma produced an EPS (EPS-I) composed of equimolar amounts of glucose and galactose that was lacking in the mutants. Phenotypic analysis revealed that the mutants had an increased propensity to form a biofilm on glass surfaces, colonized mouse lung and trachea efficiently, but had a decreased association with the A549 lung cell line. Confounding the interpretation of these results is the observation that the mutants missing EPS-I had an eightfold overproduction of an apparent second EPS (EPS-II) containing N -acetylglucosamine.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules.

We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl alpha-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II. (J Histochem Cytochem 47:481-488, 1999)     

Histochemical localization of galactose-containing glycoconjugates in sensory neurons and their processes in the central and peripheral nervous system of the rat

We studied the distribution of sugar residues in the oligosaccharide chains of complex carbohydrates in tissue sections of rat spinal cord, brainstem, and sensory ganglia using twelve lectin-horseradish peroxidase conjugates. Glycoconjugates containing terminal galactose residues were localized apparently in the Golgi apparatus in a population of predominantly small B-type neurons in spinal and trigeminal ganglia. Large A-type neurons rarely showed reactivity with galactose-binding lectins. A cells stained for glycoconjugates with N-glycosidically linked oligosaccharides and glycogen. The central and peripheral processes of the small neurons, mostly unmyelinated C fibers in sensory roots and spinal nerves, contained an abundance of glycoconjugates with terminal alpha-galactose residues. The central projections and terminals of small to medium-sized primary sensory neurons in the spinal and trigeminal ganglia were visualized in Lissauer's tract and the substantia gelatinosa in the spinal cord, and in the spinal trigeminal tract and the nucleus trigeminus in the lower medulla with lectins specific for terminal alpha-galactose residues. In addition, fibers of the solitary system and the area postrema were reactive with these lectins. The peripheral and central nervous system elements with affinity for galactopyranosyl-specific lectins correspond in distribution with neuroanatomical regions thought to be involved in the transmission and relay of somatic and visceral afferent inputs such as pain and temperature. Such specific localization of a glycosubstance to a distinct subpopulation of neurons and their peripheral and central processes suggests that the particular glycoconjugate may be of physiological significance.     

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase--PNA/SBA and galactose oxidase--neuraminidase--PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins. This revised version was published online in November 2006 with corrections to the Cover Date.     

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.     

The galactose-binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands prepared by chemical modification of glycopeptides containing triantennary N-linked oligosaccharides.

A glycopeptide containing a triantennary N-linked oligosaccharide from fetuin was modified by a series of chemical and enzymic reactions to afford a reagent that contained a terminal residue of 6-(N-methylamino)-6-deoxy-D-galactose on one branch of the triantennary structure and terminal galactose residues on the other two branches. Binding assays and gel filtration experiments showed that this modified glycopeptide could bind to the sugar-binding sites of ricin. The ligand was activated at the 6-(N-methylamino)-6-deoxy-D-galactose residue by reaction with cyanuric chloride. The resulting dichlorotriazine derivative of the ligand reacts with ricin, forming a stable covalent linkage. The reaction was confined to the B-chain and was inhibited by lactose. Bovine serum albumin and ovalbumin were not modified by the activated ligand under similar conditions, and we conclude, therefore, that the reaction of the ligand with ricin B-chain was dependent upon specific binding to sugar-binding sites. Ricin that had its galactose-binding sites blocked by the covalent reaction with the activated ligand was purified by affinity chromatography. The major species in this fraction was found to contain 2 covalently linked ligands per ricin B-chain, while a minor species contained 3 ligands per B-chain. The cytotoxicity of blocked ricin was at least 1000-fold less than that of native ricin for cultured cells in vitro, even though the activity of the A-chain in a cell-free system was equal to that from native ricin. Modified ricin that contained only 1 covalently linked ligand was also purified. This fraction retained an ability to bind to galactose affinity columns, although with a lower affinity than ricin, and was only 5- to 20-fold less cytotoxic than native ricin.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Purification and characterization of rat liver alpha-N-acetylglucosaminyl phosphodiesterase

In an earlier report we described the identification of an alpha-N-acetylglucosaminyl phosphodiesterase that is capable of cleaving the outer phosphodiester-linked alpha-N-acetylglucosamine residues present on the high mannose oligosaccharides of newly synthesized lysosomal enzymes (Varki, A., and Kornfeld, S. (1980) J. Biol. Chem. 255, 8398-8401). We have now purified this enzyme 1800-fold with a 24% yield from rat liver, using subcellular fractionation, differential extraction with Triton X-10, DEAE-cellulose chromatography, heparin-Sepharose chromatography, concanavalin A-Sepharose affinity chromatography, and gel filtration on Sephacryl S-300. The purified preparation is free of lysosomal alpha-N-acetylglucosaminidase. The enzyme exhibited a single form on both the ion exchange and gel filtration steps. It has a broad pH optimum between 6.0-8.0 and is unaffected by divalent cations or reducing agents. The enzyme cleaves alpha-N-acetylglucosamine residues from five different locations on the high mannose oligosaccharide. In the case of molecules with one phosphodiester, the rate of cleavage is not affected by the size of the underlying oligosaccharide or the presence or absence of an asparagine-linked peptide. Molecules with two phosphodiesters are cleaved in a nonrandom manner. The enzyme has no activity toward p-nitrophenyl-alpha-N-acetylglucosamine but is capable of cleaving phosphodiester-linked N-acetylglucosamine in molecules such as UDP-N-acetylglucosamine, indicating that it can only hydrolyze N-acetylglucosamine residues that are alpha-linked to a phosphate group.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Characterization of the glycoconjugates of boar testis and epididymis

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.     

Evaluation of different alpha-Galactosyl glycoconjugates for use in xenotransplantation

Porcine organs are rapidly rejected after transplantation into primate recipients due to the presence of preexisting immunoglobulins that bind to terminal galactose alpha1,3 galactose residues (alpha-galactosyl) present on porcine glycoproteins and glycolipids. Currently available immunosuppressive reagents have been largely ineffective at controlling the synthesis of these anti-Gal antibodies. Nonantigenic hapten polymers have been shown to be effective materials for blocking humoral immune responses in various model systems. We have developed a series of alpha-galactosyl glycoconjugate polymers and tested their ability to block anti-Gal antibody binding in vitro and in vivo. A galactose alpha1,3 galactose beta 1,4 GlcNAc trisaccharide free acid (TRFA) with a hexanoic acid spacer, containing five methylene groups and a carboxylic acid, was produced and coupled to a variety of polymeric backbones including dextran, branched poly(ethylene glycol) (PEG), and poly-L-lysine. The ability of monomeric TRFA and the alpha-galactosyl conjugates to block anti-Gal IgG and IgM binding was determined using a competition ELISA assay on defined HSA-Gal glycoconjugates and porcine microvascular endothelial cell substrates. We show that branched PEG carriers, with a TRFA sugar attached to each branch, exhibit enhanced antibody blocking ability compared to TRFA, but at higher target antigen densities these simple PEG conjugates are no more effective then an equivalent amount of TRFA in blocking anti-Gal IgM antibody interactions. In contrast, polymers of the branched PEG conjugates and linear conjugates made using dextran and poly-L-lysine were 2000 to 70000-fold more effective inhibitors of anti-Gal antibodies. In a study using nonhuman primates, a single dose infusion of polymeric PEG or dextran glycoconjugates dramatically reduced the level of circulating anti-Gal antibodies in cynomologus monkeys for at least 72 h. Glycoconjugates similar to these might be useful both to block anti-Gal interactions in vivo and to specifically control the induced anti-Gal immune response.     

Endocytic membrane traffic to the Golgi apparatus in a regulated secretory cell line

We have established a ricin-resistant glycosylation-defective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. The strategy employed in this study is a modification of that used previously (Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628) to demonstrate transport of the cation-independent and -dependent mannose 6-phosphate receptors from the cell surface to the trans-Golgi network in nonsecretory cell types. In ricin-resistant PC12 cells, radiolabeled galactose was incorporated enzymatically into surface glycoconjugates, primarily glycoproteins. Resistance to beta-galactosidase was acquired upon reculture at 37 degrees C due to further terminal glycosylation of the galactose residues. Treatment of N-linked oligosaccharides isolated from recultured cells with a variety of glycosidases in conjunction with beta-galactosidase demonstrated the addition of sialic acid N-acetylglucosamine and fucose residues to the galactose residues in recultured cells. Resistance to beta-galactosidase was not acquired in cells recultured at 19 degrees C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (less than 1%) after 6 h of reculture, approximately 10% of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. Analysis of total labeled versus beta-galactosidase-resistant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that endocytic traffic to the site of glycosylation activity in mutant PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts.     

A comparison of lectin binding in rat and human peripheral nerve

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.     

Ligand binding characteristics of the major mistletoe lectin.

Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand.     

CHOLINERGIC SITES IN SKELETAL MUSCLE: INTERACTION WITH CONCANAVALIN A

Abstract– The interaction of normal and denervated skeletal muscle cholinergic sites with the lectin concanavalin A and concanavalin A-Sepharose are detailed. Concanavalin A blocks the binding of ¹²⁵I-α-bungarotoxin to both the high and low affinity sets of cholinergic sites described previously. The characteristics of the block of ¹²⁵I-α-bungarotoxin binding to the high affinity set (acetylcholine receptor) is not competitive. The data suggest that the concanavalin binds multivalently to the macro-molecular complex containing the ACh receptor site and sterically prevents the α-bungarotoxin binding. The interaction of both sets of cholinergic sites with concanavalin A-Sepharose was also studied. The macromolecule(s) containing both the high and low affinity sets of sites bind to the concanavalin A-Sepharose. The data indicate a multivalent association with the affinity resin. Following the affinity procedure, a partial purification in both sets of sites is effected. The equilibrium binding of ¹²⁵I-diiodo-α-bungarotoxin to the preparations from the affinity procedure (both normal and denervated muscle) was examined. The K_D of the α-bungarotoxin binding to the high affinity sets of sites (acetylcholine receptor) in both normal and denervated preparations changes from ∼10⁻⁹mol/l to ∼ 10⁻¹⁰ mol/l following purification. No change in the K_D of the α-bungarotoxin binding to the low affinity set of sites was observed following purification. The ¹²⁵l-α-bungarotoxin binding to the partially purified acetylcholine receptor was blocked by unlabelled α-bungarotoxin, concanavalin A, d-tubocurarine and carbamylcholine.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins.

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with alpha-methylmannoside, constitute about 25--30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chloride columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with alpha-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.