Lectin Analysis of Trypanosoma congolense Bloodstream Trypomastigote and Culture Procyclic Surface Saccharides by Agglutination and Electron Microscopic Technics*

SYNOPSIS. Living, intact bloodstream trypomastigotes and culture procyclic forms of Trypanosoma congolense were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose binding protein (FBP). Similar experiments were conducted with living bloodstream and culture forms treated with trypsin or dextranase. Parasites were incubated for 30 min at 25 C in various concentrations of each lectin, then examined for agglutination by dark-field microscopy. Control preparations consisted of parasites incubated alone or with 0.5 M of the specific competing sugar, with or without the corresponding lectin. Electron-microscopic localization of lectin binding sites on the surface of intact and dextranase-treated bloodstream and intact culture forms was accomplished with Con A, reacted with horseradish peroxidase (HRP) and then diaminobenzidine (DAB). In addition, FBP and SBA were coupled to HRP, then utilized for the localization of binding saccharides on the surface of blood-stream forms by the DAB technic. Similar studies were conducted with culture procyclics incubated with WGA-, SBA, PP- or FBP-HRP conjugates and then reacted with DAB. Controls were utilized to confirm the sugar specificity of all positive reactions. Intact living bloodstream forms were agglutinated in a concentration-dependent manner with all the lectins tested. Agglutination levels were scored as Con A > FBP > WGA = PP = SBA. Sugars resembling α-D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and α-L-fucose are evidently present on the surface of the parasites. No agglutination was noted in any control preparations. Identical lectin-induced agglutinations were obtained with trypsin- or dextranase-treated bloodstream forms. Trypsin disrupted but did not entirely remove the surface coat of bloodstream forms, while dextranase did not alter the ultrastructure of the parasites. Con A-, SBA- and FBP-binding saccharides were distributed uniformly on the surface coat of intact bloodstream forms; a similar distribution of Con A receptors was noted also on the surface of dextranase-treated cells. No lectin-binding saccharides were visualized by electron microscopy on any control preparations. Intact, trypsin- or dextranasetreated, procyclics were agglutinated in a concentration-dependent fashion by Con A and WGA, but not by the other lectins tested. Control preparations did not agglutinate and the enzymes did not affect the ultrastructure of the parasites. Con A- and WGA-specifically binding saccharides were uniformly distributed on intact procyclics and control preparations were lectin-negative. Thus, T. congolense procyclics retained surface saccharides resembling α-D-mannose and N-acetyl-D-glucosamine but lost sugars resembling N-acetyl-D-galactosamine (or D-galactose) and α-L-fucose. The failure of dextranase to remove the lectin-binding saccharides from the surface of bloodstream and procyclic forms suggests that α-1,6-glucan bonds do not link these carbohydrates. The results are contrasted with lectin research on other trypanosome species and discussed with relation to the biology of T. congolense.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. II. Lectin and Immunologic Studies*

SYNOPSIS. Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing α-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and α-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with α-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with α-1,4 or repetitive α-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.