The effect of glutaraldehyde fixation on lectin-mediated agglutination of murine leukaemia (GRSL) cells was investigated using 2 assay methods which differed in the shear forces to which the agglutinated cells were subjected. First, lectin and cells were allowed to interact under conditions in which shear forces were minimized and the degree of agglutination was evaluated microscopically by the appearance and size of the cell aggregates. This assay demonstrated that concanavalin A (con A)-, wheat germ agglutinin (WGA)- or Ricinus communis agglutinin I (RCAI)-mediated cytoagglutination was unaffected (WGA and RCAI) or somewhat enhanced (con A) by prior fixation of the cells with glutaraldehyde. Secondly, an electronic particle counter was used to measure the disappearance of single cells and concomitant appearance of cell aggregates as a function of the lectin concentration. In this assay, in which the aggregated cells are subjected to significant shear forces during dilution and cell counting, agglutination of GRSL cells by each of the 3 lectins was drastically inhibited by prior fixation of the cells with glutaraldehyde. This assay also demonstrated enhanced nonlectin-induced cell aggregation after fixation. In both cytoagglutination assays about the same lectin concentration was required for threshold agglutination of unfixed cells. Comparatively, the results of the 2 cytoagglutination assays indicate that a fraction of the lectin-mediated bonds between unfixed cells is shear resistant and that fixation of the cells either weakens these bonds or inhibits their formation. Morphologically, cells prefixed with glutaraldehyde were sperical at all lectin concentrations, with a continuous dense distribution of cell surface-bound con A, labelled directly with haemocyanin or indirectly using the peroxidase-diaminobenzidine reaction. Unfixed cells showed angular and toadstool-shaped deformations, especially at the highest lectin concentrations, the agglutinating surfaces being flattened against each other over extended areas. The distribution of con A label was continuous and dense between the apposed surfaces and discontinuous on free surfaces. In the presence of con A the free surfaces of prefixed cells exhibited more microvilli than the surfaces of non-prefixed cells. These results favour the view that fixation prevents the formation of shear-resistant, lectin-mediated bonds between cells, not by restricting the lateral mobility of lectin receptors, but by impairing the apposition of rigid cell surfaces.

Agglutination↗

PubMed1976

Effect of neuraminidase and papain treatment on lectin-induced agglutination of Novikoff tumor cells and assay of lectin receptor activity of the glycopeptides released from the cell surface by papain.

Lectins, plant proteins that bind specific saccharide determinants, have been utilized to examine the effect of neuraminidase digestion on the structure and/or expression of oligosaccharide moieties present at the periphery of Novikoff ascites hepatoma cells. Five lectins were utilized: concanavalin A (Con A), specific for alpha-D-manno- or alpha-D-glucopyranosyl residues; wheat germ agglutinin, specific for 2-acetamido-2-deoxy-D-glucopyranosyl residues; Ricinus communis agglutinin I (RCAI), specific for D-glucopyranosyl residues; R. communis agglutinin II (RCAII), specific for D-galacto- or 2-acetamido-2-deoxy-D-galactopyranosyl residues; and soybean agglutinin, specific for 2-acetamido-2-deoxy-D-galactopyranosyl residues. Neuraminidase treatment of Novikoff cells did not alter their agglutination by Con A or wheat germ agglutinin. Similar treatment produced only a 2-fold increase in their agglutination by RCAI but a 12-fold increase in their agglutination by RCAII, indicating that 2-acetamido-2-deoxy-D-galactopyranosyl residues become expressed upon neuraminidase treatment. This conclusion was confirmed by the observation that neuraminidase-treated Novikoff cells acquired agglutinability by soybean agglutinin. Binding studies using ferritin-conjugated RCAII indicated that neuraminidase treatment exposed cryptic cell surface receptors for RCAII. To ascertain the role of cell surface glycoproteins in lectin-induced agglutination of Novikoff cells, glycopeptides cleaved from the cell surface by papain were assayed for lectin receptor activity. The cell surface glycopeptides exhibited receptor activity for Con A, wheat germ agglutinin and RCAI but not for RCAII and soybean agglutinin. A cell surface macrosialoglycopeptide fraction, resolved by gel filtration and ion-exchange chromatography, possessed a major portion of the Con A and RCAI receptor activity.

Agglutination↗

PubMed1975