Glycoconjugate Residues Research Articles

High and low molecular weight tracers for the electron microscopical detection of sialoglycoconjugates.

Hydrazide-derivative tracers of different molecular weights have been synthesized for use in the electron microscopical detection of sodium periodate-oxidized sialyl residues of glycoconjugates in various tissues and cells. Haemundecapeptide hydrazide, horseradish peroxidase hydrazide, and Limulus polyphemus haemocyanin hydrazide were obtained by coupling adipic acid dihydrazide to the tracers with the aid of water-soluble carbodiimide. The enzymatic tracers thus prepared retained their peroxidatic activity. On conversion to the hydrazide derivative, the haemocyanin molecule dissociated into its hexameric subunits. In order to test by transmission electron microscopy the ability of the conjugates to bind to the sialoglycoconjugates of endothelial cell surfaces, each tracer was perfused in situ into rat pancreatic vasculature previously oxidized with 1 mM sodium periodate. The three tracers characteristically labelled the various microdomains of the luminal cell coat of the capillary endothelial cell. The electron opacity of the haemocyanin subunits allowed their easy detection when bound to the cell surface or to components of the extracellular matrix. The bound markers were not displaced by a high ionic strength buffer, and did not label desialylated cell surfaces. These results indicate that the three hydrazide-derivative tracers may be useful tools for the electron microscopical detection of cellular and extracellular sialoglycoconjugates.

Analysis of neutral sugars as dinitrophenyl-hydrazones by high-performance liquid chromatography

A new method for the separation and determination of neutral sugars as 2,4-dinitrophenyl derivatives is presented. After a simple derivatization procedure at 65°C for 90 min and purification of the reaction mixture from excess of reagents, the sugar derivatives were separated on a LiChrosorb Si 100 column using methanolwater in chloroform as the eluent and UV detection at 352 nm. The yield of the products is affected by several parameters. However, when the proposed conditions are closely adhered to an excellent precision is obtained. Calibration graphs are linear in the range of 0.05–3.35 nmol of each sugar injected. The method was used for the determination of neutral sugars normally found in glycoconjugates, namely fucose, xylose, mannose, galactose and glucose. Hexosamines, uronic acids, alditoles and amino acids do not interfere with the analysis. Thus, the derivatization procedure can be applied directly to unfractionated acid hydrolysate residues of glycoconjugates.

Lectin-binding pattern in parotid acinar cells

The localization of complex carbohydrates in the Golgi apparatus, secretory granules and plasmalemma of mouse parotid acinar cells was studied using the fracture-labelling method. The hexose residues of glycoconjugates were identified using ferritin conjugated with Wheat Germ Agglutinin (WGA-), Ricinnus Communis Agglutinin II (RCA-II-), Phaseolus Vulgaris Agglutinin (PHA-) and Limulus Polyphemus Agglutinin (LPA-). We found that the fracture-labelling method allows not only the labelling of membrane faces but also analysis of the compartment's content that is exposed during the fracturing of the tissue. Our results revealed differences in the hexose residues located in the Golgi apparatus, secretory granules and the apical and lateral plasmalemma. Numerous binding sites for WGA-, PHA- and RCA-II-ferritin were demonstrable in the Golgi apparatus. In secretory granules, the WGA- and RCA-II-ferritin binding sites were most numerous, while LPA-ferritin binding sites were very rare. The density of the binding sites for PHA-ferritin showed considerable variation in secretory granules. The apical plasmalemma exhibited a high density of binding sites for all of the lectins used. In the lateral plasmalemma, LPA-ferritin was not bound, and there were fewer binding sites for WGA-, RCA-II- and PHA-ferritin.

The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells

Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.

Identification of Lectin-like Substances Recognizing Galactosyl Residues of Glycoconjugates on the Plasma Membrane of Marrow Sinus Endothelium

Plasma membrane components capable of specific binding to the sugar residues of glycoproteins have recently been identified in several cell systems and implicated in the recognition and specific uptake of soluble or membrane-bound glycoproteins. Because the endothelium of bone marrow sinuses is the site of massive cellular and molecular traffic that is often specific, we studied the surface of sinus endothelium for the presence of sugar-recognizing systems. Neoglycoprotein probes were synthesized by covalently binding bovine serum albumin (BSA) to activated pyrannose form of galactose, fucose, or mannose. The probe was then labeled with colloidal gold. Galactosyl-BSA gold bound to endothelial membrane at 4 degrees C and was internalized at 37 degrees C. Both the binding and the internalization were inhibited in the presence of excess unlabeled galactosyl-BSA. Gold-labeled mannosyl-BSA and fucosyl-BSA did not bind to the endothelium. Nor did BSA-gold without galactosyl residues bind to endothelial membrane, confirming that galactosyl moiety was responsible for the binding. The uptake of galactosyl-BSA was further confirmed by perfusion of 125I-galactosyl-BSA into the abdominal aorta. Small but highly specific uptake was noted in tibias and femurs. These data provide evidence for the presence of a lectin-like substance on the luminal surface of marrow endothelial membrane capable of specific interaction with galactosyl residues of circulating and cell-bound glycoproteins and may provide a mechanism for specific recognition of these glycoproteins.

Identification of lectin-like substances recognizing galactosyl residues of glycoconjugates on the plasma membrane of marrow sinus endothelium

Plasma membrane components capable of specific binding to the sugar residues of glycoproteins have recently been identified in several cell systems and implicated in the recognition and specific uptake of soluble or membrane-bound glycoproteins. Because the endothelium of bone marrow sinuses is the site of massive cellular and molecular traffic that is often specific, we studied the surface of sinus endothelium for the presence of sugar-recognizing systems. Neoglycoprotein probes were synthesized by covalently binding bovine serum albumin (BSA) to activated pyrannose form of galactose, fucose, or mannose. The probe was then labeled with colloidal gold. Galactosyl- BSA gold bound to endothelial membrane at 4 degrees C and was internalized at 37 degrees C. Both the binding and the internalization were inhibited in the presence of excess unlabeled galactosyl-BSA. Gold- labeled mannosyl-BSA and fucosyl-BSA did not bind to the endothelium. Nor did BSA-gold without galactosyl residues bind to endothelial membrane, confirming that galactosyl moiety was responsible for the binding. The uptake of galactosyl-BSA was further confirmed by perfusion of 125I-galactosyl-BSA into the abdominal aorta. Small but highly specific uptake was noted in tibias and femurs. These data provide evidence for the presence of a lectin-like substance on the luminal surface of marrow endothelial membrane capable of specific interaction with galactosyl residues of circulating and cell-bound glycoproteins and may provide a mechanism for specific recognition of these glycoproteins.

Ultrastructural methods for localizing complex carbohydrates

Carbohydrate-rich macromolecules in mammalian tissue fall into two main groups, glycoproteins and glycosaminoglycans. Although glycoproteins occur mainly in epithelial cells and glycosaminoglycans in connective tissue interstices, exceptions are known. Chemical constituents that are characteristic of glycoproteins and provide a basis for their selective demonstration cytochemically include the sulfate ester, presumably bound to an internal sugar, the carboxyl group of terminal sialic acid, and the vicinal glycol of hexose (galactose or mannose) and deoxyhexose (fucose). Characteristic chemical components of glycosaminoglycans that are detectable cytochemically include the sulfate ester, usually linked to an aminosugar, and the carboxyl group of glucuronic or iduronic acid. Individual sugars of glycoconjugates, particularly those in a terminal positions, are also identifiable cytochemically. Cytochemical methods are available to detect specifically one or more of the moieties characteristic of glycoproteins or glycosaminoglycans at the electron microscopic level. The sulfate esters can be localized specifically by pre-embedment staining with the high-iron diamine (HID) method. Dialyzed iron (DI) and cationized ferritin stain both carboxyl groups and sulfate esters when similarly applied before embedment. In addition, these cationic reagents can be utilized to stain at least some sulfated glycocon-jugates in post-embedment procedures carried out on ultrathin sections. Ruthenium red in the fixative solution localizes glycoconjugate and possibly other constituents on the cell surface or in the extracellular space. The periodic acid-thiocarbohydrazide-silver proteinate (PA-T-SP) method and the tannic acid-uranyl acetate sequence carried out after embedment on epoxy thin sections demonstrate sugar residues of glycoconjugates, extending light microscopic periodic acid-Schiff (PAS) staining to the ultrastructural level. Further specificity can be achieved by pre- or post-embedment staining with conjugates of horseradish peroxidase or ferritin to a lectin with affinity for a specific sugar in a complex carbohydrate and by enzymatic digestion with specific glycosidase to decrease or increase staining with the above mentioned methods. DI-positive, HID-negative sites can be interpreted as containing nonsulfated, carboxylated complex carbohydrate, and HID-positive sites as containing sulfated mucosubstance. DI- or HID-positive sites lacking PA-T-SP reactivity, as a rule, consist of glycosaminoglycans, but infrequently contain periodate-negative glycoprotein. On the other hand, PA-T-SP staining and DI or HID non-reactivity evidence the presence of neutral glycoprotein, whereas PA-T-SP positivity plus basophilia indicates acidic glycoprotein. Examples of applications of these methods are presented. The applications to normal tissue provide a basis for comparison with diseased specimens and point to the potential of carbohydrate histochemistry for investigating and diagnosing pathologic change.