Specificity of C-glycoside complexation by mannose/glucose specific lectins.

The binding of the mannose/glucose specific lectins from Canavalia ensiformis (concanavalin A) and Dioclea grandiflora to a series of C-glucosides were studied by titration microcalorimetry and fluorescence anisotropy titration. These closely related lectins share a specificity for the trimannoside methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, and are a useful model system for addressing the feasibility of differentiating between lectins with overlapping carbohydrate specificities. The ligands were designed to address two issues: (1) how the recognition properties of non-hydrolyzable C-glycoside analogues compare with those of the corresponding O-glycosides and (2) the effect of presentation of more than one saccharide recognition epitope on both affinity and specificity. Both lectins bind the C-glycosides with affinities comparable to those of the O-glycoside analogues; however, the ability of both lectins to differentiate between gluco and manno diastereomers was diminished in the C-glycoside series. Bivalent norbornyl C-glycoside esters were bound by the lectin from Canavalia but only weakly by the lectin from Dioclea. In addition to binding the bivalent ligands, concanavalin A discriminated between C-2 epimers, with the manno configuration binding more tightly than the gluco. The stoichiometry of binding of the bivalent ligands to both di- and tetrameric lectin was two binding sites per ligand, rather than the expected 1:1 stoichiometry. Together, these results suggest that concanavalin A may possess more than one class of carbohydrate binding sites and that these additional sites show stereochemical discrimination similar to that of the previously identified monosaccharide binding site. The implications of these findings for possible in vivo roles of plant lectins and for the use of concanavalin A as a research tool are discussed.

Fetal bovine serum and other sera used in tissue culture increase epithelial permeability.

Fetal bovine serum (FBS) or heat-inactivated FBS (56 degrees C for 30 min, HFBS) caused a dose-dependent decrease in the transepithelial electrical resistance of an epithelial monolayer (MDCK). A saturating concentration of HFBS (30%) caused an average fall of 25 +/- 2% within 60 min. Upon removal of HFBS, the resistance returned to its starting value within 1 h. Flux studies with [3H]mannitol demonstrate that the fall in resistance is due to an increased permeability of the tight junctions. Thirty percent heat inactivated sera from goat, newborn calf, calf, bovine, and horse caused falls ranging from 26 to 47%. In contrast with the basolateral preference of human and bovine adult sera, fetal bovine and newborn calf sera elicit this response primarily by interacting with the apical surface of the epithelium. HFBS-treated monolayers show a significant increase in the condensation of F-actin at points where > or = 3 cells meet. These results demonstrate that FBS and other sera used as nutritional supplements can increase the permeability of the tight junctions of cultured epithelial cells.

Epithelial permeability factor: a serum protein that condenses actin and opens tight junctions.

An epithelial permeability factor (EPF) in human serum lowered, within 1 h, the transepithelial electrical resistance and opened the tight junctions of a cultured kidney epithelium (Madin-Darby canine kidney) when it came in contact with the basolateral surface of the kidney epithelium. Size-exclusion chromatography of serum or heat-inactivated serum resolved seven peaks of EPF activity (approximately 15, approximately 30, approximately 45, approximately 60, approximately 120, and approximately 240 kDa and greater than 240 kDa) with 65% of the activity at approximately 45, approximately 60, and approximately 120 kDa. Heat inactivation, which had no effect on total activity, caused a significant decrease in the activity at 120 kDa and an equivalent rise in activity at 45 kDa. Although acid charcoal extraction or lectin affinity chromatography did not remove activity, EPF activity was eliminated by pepsin. Heat-inactivated serum or fractions containing EPF had no effect on ZO-1 localization but did cause a dose-dependent focal condensation of the perijunctional actin ring at sites where three or more cells were in contact. These data suggest that EPF is a protein that appears to form multimers that interact with the basolateral surface of the epithelium and cause constriction of the cytoskeleton and an increase in permeability at specific sites along the tight junction.