Histochemical detection of glycogen and glycoconjugates in the inner ear with modified concanavalin A-horseradish peroxidase procedures

Summary Inner ears from neonatal and adult Mongolian gerbils were examined to determine developmental changes in the content of glycogen and glycoconjugates as shown by histochemical application of the jack bean lectin, concanavalin A (con A). Sections of fixed paraffin-embedded inner ears were stained using the con A-horseradish peroxidase sequence in conjunction with prior treatments including periodate oxidation with or without subsequent reduction and diastase digestion. In adult inner ear, brief periodate oxidation followed by reduction and con A-horseradish peroxidase staining demonstrated abundant glycogen in Deiters' cells and in fibrocytes of the spiral ligament and submacular plaque. This procedure also detected diastase-resistant glycoprotein, probably containing N-linked complex-type saccharides, in the basal and marginal regions of the tectorial membrane and in the otolithic membrane. During morphogenesis and maturation, various cochlear cells showed changes in their glycogen content possibly related to stage-specific energy requirements. Cellular glycogen storage reached adult levels by postnatal day 14. The tectorial membrane gradually acquired con A reactivity during the first postnatal week. Thus, application of modified con A staining procedures has provided further knowledge for comparison with data from previous biochemical and histochemical studies of carbohydrate-rich components in the inner ear.     

Cell Surface Carbohydrates in Tritrichomonas foetus1

The cell surface of Tritrichomonas foetus was characterized by using 18 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose, and sialic acid. The specificity of the lectin-induced cell agglutination was verified by inhibition of the agglutination with the specific sugars. By using cytochemical techniques associated with electron microscopy, carbohydrates were detected on the cell surface of T. foetus. The following techniques were used: periodic acid-thiosemicarbazide-silver proteinate, concanavalin A-horseradish peroxidase, and ruthenium red. Anionic sites were detected on the cell surface of the protozoan at pH's 1.8 and 7.2 with the use of colloidal iron hydroxide and cationized ferritin particles, respectively. The binding of colloidal iron particles, as well as the agglutination induced by the lectin from Limulus polyphemus, indicated the presence of sialic acid on the cell surface of T. foetus.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Post-embedding staining of rat gastric mucous cells with lectins

Summary The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinuscommunis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.This work was supported in part by a grant-in-aid (No. 457008) from the Ministry of Education, Science and Culture, Japan and a grant-in-aid for cancer research (55-21) from the Ministry of Health and Welfare, Japan     

Cytochemical localization of glycoconjugates in rat peritoneal mast cells during degranulation

Summary Cytochemical methods for the localization of glycoconjugates including concanavalin A-horseradish peroxidase (ConA-HRP) and dialysed iron were used to study the distribution of glycoconjugates in mast cell granules during degranulation. The ConA-HRP method revealed intense staining of discharged mast cell granules. Dialysed iron staining was seen at the granule periphery, with extruded granules exhibiting more intense staining than undischarged granules.Some of the work reported herein was performed in partial fulfillment of the requirements for a Ph.D. degree.     

Concanavalin A reactivity of vitellogenin and yolk proteins of the threespined stickleback Gasterosteus aculeatus (Teleostei)

1. Concanavalin A (con A) reactive proteins have been detected in the plasma and ovaries of the oestradiol treated Gasterosteus aculeatus. 2. Concanavalin A-horseradish peroxidase (HRP) technique applied on nitrocellulose membranes reveals that vitellogenin (Vg) is the only mannose and glucose rich glycoprotein present in the plasma of oestradiol treated sticklebacks. Stickleback Vg can be purified by con A-Sepharose chromatography. 3. Con A reactivity in the ovary changes in the course of development of the oocytes. First, the yolk vesicles, which are synthesized by the oocyte itself, become con A positive. Later, the yolk granules, which contain vitellogenin synthesized in the liver and taken up from the plasma, show a clear affinity for con A. Con A staining disappears when mannopyranoside is added. 4. No con A staining is found in the periodic acid/Schiff staining chorion.     

Concanavalin A-horseradish peroxidase bridge staining of alpha-1 glycoproteins separated by isoelectric focusing on polyacrylamide gel.

The Coomassie Blue protein stain and the periodic acid-Schiff stain for glycoproteins are compared to a new method of staining glycoproteins resolved electrophoretically. The method utilizes a Concanavalin A-horseradish peroxidase sequence to visualize selectively glycoproteins with terminal or internal mannose or terminal N-acetylglucosamine. The method applied to characterization of M and Z allele products of alpha-l-antitrypsins separated by isoelectric focusing of polyacrylamide gels slabs have revealed differences in carbohydrate content of various components that were previously undetected.     

Major Central Nervous System Myelin Glycoprotein of the African Lungfish (Protopterus dolloi) Cross-Reacts with Myelin Proteolipid Protein Antibodies, Indicating a Close Phylogenetic Relationship with Amphibians

CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti-human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A-horseradish peroxidase and bound to anti-rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti-PLP reactivity. These results agree with a concanavalin A-binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g-PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP-cross-reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.     

The reaction of coumarins with horseradish peroxidase

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.     

Effects of ovarian hormones on cell membranes in the rat uterus. III. The surface carbohydrates at the apex of the luminal epithelium

Histochemical techniques, including radioisotope histochemistry, have been used to investigate the nature of the surface carbohydrates at the apex of cells of the luminal epithelium of the rat uterus under various hormonal conditions. Binding of ruthenium red was quantitatively similar in ovariectomized rats without further treatment and in those given three daily injections of progesterone. Ruthenium red binding was significantly lower after 3 days treatment with estradiol, and also after 3 days treatment with progesterone with an additional dose of estradiol on day 3, a regime known to produce an epithelium receptive to the implanting blastocyst. Binding of concanavalin A (con A), whether studied by electron microscope histochemistry after incubation of tissue with con A-horseradish peroxidase, or by light microscope autoradiography after incubation with 3H-con A, was not statistically different in any of the four groups of rats. The results with ruthenium red show a reduction in net negative charge of the carbohydrates on the apical cell membrane in conditions permitting implantation: this change is not due to variations in the amounts of the neutral carbohydrates, mannose and glucose, as demonstrated by con A.     

Effects of ovarian hormones on cell membranes in the rat uterus

Histochemical techniques, including radioisotope histochemistry, have been used to investigate the nature of the surface carbohydrates at the apex of cells of the luminal epithelium of the rat uterus under various hormonal conditions. Binding of ruthenium red was quantitatively similar in ovariectomized rats without further treatment and in those given three daily injections of progesterone. Ruthenium red binding was significantly lower after 3 days treatment with estradiol, and also after 3 days treatment with progesterone with an additional dose of estradiol on day 3, a regime known to produce an epithelium receptive to the implanting blastocyst. Binding of concanavalin A (con A), whether studied by electron microscope histochemistry after incubation of tissue with con A-horseradish peroxidase, or by light microscope autoradiography after incubation with³H-con A, was not statistically different in any of the four groups of rats. The results with ruthenium red show a reduction in net negative charge of the carbohydrates on the apical cell membrane in conditions permitting implantation: this change is not due to variations in the amounts of the neutral carbohydrates, mannose and glucose, as demonstrated by con A.     

Evidence for a glycoconjugate form of glutathione S-transferase pI.

The anionic form of glutathione S-transferase from human (GST pi) and rat (GST Yp) sources has been shown to exist in multiple forms which have similar molecular weights but different isoelectric points (pIs). Treatment with endoglycosidase H caused the acidic forms of GST Yp to be converted to proteins with more basic pIs as compared to the untreated control mixtures, suggesting that an N-linked mannose moiety containing acidic residues had been removed. Inability to detect these carbohydrates by techniques requiring unsubstituted vicinal hydroxyls further suggested acidic substitutions on the sugar moiety. GST pi/Yp carbohydrate modifications were also identified by differential staining procedures. These data represent the first indication that glycosylation of GST can occur. Additionally, this may offer an explanation for the often seen microheterogeneity within a class of GST isozymes.     

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.     

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.     

A Co(III) derivative of concanavalin A

Co(III) has been stoichiometrically incorporated into jack bean concanavalin A. The Co(III) protein still possesses a binding site for an additional divalent transition metal ion which together with Ca(II) can induce the sugar binding ability. No H₂O₂ oxidation of Co(II) occurs with demetallized concanavalin A activated with Ca(II) and Co(II) unless Co(II) is present in a stoichiometric excess. Evidence is presented to indicate that kinetically stable Co(III) is bound to a completely different location than the thermodynamically stable Co(II) protein site.     

Synthesis of a novel class of glycocluster with a cyclic α-(1→6)-octaglucoside as a scaffold and their binding abilities to concanavalin A

The synthesis of small glycoclusters with high affinity toward lectins is one of the important subjects in glycotechnology. Although cyclic α-(1→6)-d-octaglucoside (CI8) is an attractive scaffold on which to put glycosyl pendants, the compound has only secondary hydroxyl groups, which are relatively unreactive for substitution reactions. The oxidation of the vicinal diols of CI8 and reductive amination of the resultant dialdehydes with 2-aminoethyl mannoside gave mannose-CI8 conjugates with a variety of average mannose incorporation numbers (2-7). The average numbers were deduced from MALDI-TOF mass and ¹H NMR spectroscopy. The binding ability of mannose-CI8 conjugates to concanavalin A increased with the increasing numbers of average mannose incorporation, reaching a plateau at tetravalence, as estimated from a latex bead-based agglutination lectin assay. Toxicity tests demonstrated the biocompatibility of mannose-CI8 conjugates.     

Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A-horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.     

The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase.

The plant lectin concanavalin A (Con A) possesses a remarkably specific capacity to bind primarily α-d-mannose or α-d-glucose sugar residues on macromolecules (cf. 1). The multivalent Con A will bind to carbohydrates on cell surfaces, and free binding sites on the attached Con A will bind to horseradish peroxidase which is a glycoprotein (2). Since peroxidase may be visualized by reaction with diaminobenzidine (3), it has been possible using this method to specifically “stain” carbohydrate residues on cell surface macromolecules (2, 4). The same principles for staining cell surfaces should apply to “staining” glycoproteins separated by polyacrylamide electrophoresis. In this paper, we examine the staining of glycoproteins in sciatic nerve by a Con A-peroxidase labeling technique. The method is more sensitive for mannose or glucose containing glycoproteins than the periodic acid-Schiff's (PAS) method commonly used.     

A monosaccharide-modified peptide phage library for screening of ligands to carbohydrate-binding proteins

A monosaccharide-modified β-loop peptide library displayed on phage has been constructed and used for the screening of glycopeptide ligands against a carbohydrate-binding protein. The β-loop peptide library was designed and modified with a mannose derivative on phage. The glycopeptide ligands to concanavalin A (ConA), a mannose-binding protein, were obtained from the mannose-modified peptide phage library. The amino acids neighboring the mannose unit of glycopeptides not only reinforced the binding affinity but also gave diverse binding characteristics.     

Hydrophobic anion activation of human liver chi chi alcohol dehydrogenase

Class III alcohol dehydrogenase (chi chi-ADH) from human liver binds both ethanol and acetaldehyde so poorly that their Km values cannot be determined, even at ethanol concentrations up to 3 M. However, long-chain carboxylates, e.g., pentanoate, octanoate, deoxycholate, and other anions, substantially enhance the binding of ethanol and other substrates and hence the activity of class III ADH up to 30-fold. Thus, in the presence of 1 mM octanoate, ethanol displays Michaelis-Menten kinetics. The degree of activation depends on the size both of the substrate and of the activator; generally, longer, negatively charged activators result in greater activation. At pH 10, the activator binds to the E-NAD+ form of the enzyme to potentiate substrate binding. Pentanoate activates methylcrotyl alcohol oxidation and methylcrotyl aldehyde reduction 14- and 30-fold, respectively. Such enhancements of both oxidation and reduction are specific for class III ADH; neither class I nor class II shows this effect. The implications as to the nature of the physiological substrate(s) of class III ADH are discussed in light of the recent finding that this ADH and glutathione-dependent formaldehyde dehydrogenase are identical. A new rapid purification procedure for chi chi-ADH is presented.