Binding determinants of the sialic acid-specific lectin from the slug Limax flavus.

The specific structural features of 24 N-acetylneuraminic acid derivatives required for the high affinity interaction of sialoglycoconjugates with the sialic acid-specific lectin from the slug Limax flavus were studied by hapten inhibition of precipitation. These results provide insight regarding the structure of the binding pocket for N-acetylneuraminic acid that exists in L. flavus agglutinin (LFA). The alpha-anomer of sialic acid is a very important factor in binding to the slug lectin. The carboxylic acid group makes only a moderate contribution to binding, since modifications of the carboxylic group decrease binding approximately 5-fold. Modification or removal of the hydroxyl group on carbon 4 does not affect binding. However, when the C4 epimer was tested, there was a dramatic decrease in binding, suggesting that whereas the equatorial hydroxyl at C4 does not contribute to binding, the introduction of an axial hydroxyl group at C4 sterically hinders the binding interaction. The substituent on the 5-amino group occupies an important role in binding of Neu5Ac to LFA as well. When the acetyl is modified by the addition of a hydroxyl group to yield the N-glycolyl derivative, we observed a 20-fold decrease, while the removal of the methyl to form the N-formyl derivative resulted in a 50-fold decrease. The 5-amino derivative was the poorest inhibitor of all compounds examined, indicating a critical role for the N-acetyl group in high affinity binding to LFA. The glyceryl tail also appears to be critical for binding inasmuch as acetylation of the C9 hydroxyl group or periodate cleavage of carbons 8 and 9 resulted in a 20- to 50-fold decrease in binding. The equilibrium constant (K(a)) for binding of Neu5Ac to LFA is 3.8 x 10(4) M-1, with a single binding site (n = 0.85) per monomer.