Binding of kidney bean (Phaseolus vulgaris) isolectins to differentiated human colon carcinoma Caco-2 cells and their effect on cellular metabolism

Abstract

The binding of Phaseolus vulgaris (PHA) isolectins L4 and E4 to the brush border membrane of differentiated Caco-2 cells was studied and the impact on cellular metabolism and microvilli was assessed. Computer analysis of the data based on binding experiments with peroxidase conjugated isolectins gave mean (SD) values for maximal binding of 2540 (151).10-9 M for PHA-L4 and 2104 (140). 10-9 M for PHA-E4 per mg of brush border membrane protein. The dissociation constants for L4 and E4 binding were 4.3 (1.4).10-6 M and M (0.8).10-6 M, respectively. Incubation of differentiated Caco-2 cells for 30 minutes with ferritin conjugated PHA isolectins showed that, as indicated by the number of ferritin particles, PHA-E4 bound to the microvilli to a greater extent than PHA-L4. Ferritin particles were also localised intracellularly over endocytotic invaginations and vesicles. After incubation for 48 hours with PHA-L4 or PHA-E4, the relative incorporation of precursors for DNA, RNA, and (glyco)protein synthesis into the trichloroacetic acid insoluble fraction of the Caco-2 cells was determined. Both isolectins stimulated the incorporation of thymidine and glucosamine, but neither PHA-L4 nor PHA-E4 were able to influence the incorporation of undine. With respect to fucose, methionine, and N-acetyl mannosamine, the stimulatory effect remained confined to PHA-E4. Since PHA-L4 and PHA-E4 were tested at the same concentrations, PHA-E4 is more effective than PHA-L4. The changes in the uptake of radioactive precursors were lost after heat inactivation of PHA-E4. Compared with control and PHA-L4 incubated Caco-2 cells, the microvilli of PHA-E4 incubated cells were shortened significantly (p<0.01).