ESSENTIAL AMINO-ACIDS INVOLVED IN GLUCAN-DEPENDENT AGGREGATION OF STREPTOCOCCUS-SOBRINUS

The active site of the glucan-binding lectin (or agglutinin) (GBL) of Streptococcus sobrinus was probed by specific amino acid modifying rea gents. Reagents specific for carboxylates, imidazolium, phenolic, and lysyl residues inactivated the cell bound GBL, whereas agents specific for sulfhydryl, disulfide, and guanidinium groups had no effect on th e lectin. A low molecular weight alpha-(1 --> 6)-glucan provided parti al protection against the reagents which inactivated the protein, wher eas an alpha-(1 --> 4)-glucan, incapable of complexing with the lectin , afforded no protection. A reagent specific for tryptophan, 2-hydroxy -5-nitrobenzyl bromide (HNB) did not cause a loss of GBL activity, alt hough N-bromosuccinimide, a reagent capable of oxidizing tryptophan an d less selective than HNB, was a very effective inhibitor of the gluca n-dependent cellular aggregation. In the latter case, alpha-(1 --> 6)- glucan did not protect. Hydroxylamine partially restored the loss of l ectin activity due to treatment of the cells with N-acetylimidazole (h ighly specific for tyrosine), glycine methyl ester plus water-soluble carbodiimide (specific for carboxylates), and diethylpyrocarbonate (sp ecific for histidine). Because the soluble form of GBL rapidly loses a ctivity when purified, it was necessary to perform the chemical modifi cation of the amino acid side chains employing the cell-bound form of the lectin. Because specific ligand [alpha-(1 --> 6)-glucan] protected against the inactivation of the agglutinin by selected reagents and b ecause lectin activity could be restored in some cases, it was possibl e to identify likely essential amino acid residues needed for glucan b inding. The results, taken together, suggest that aspartic (and/or glu tamic) acid, histidine, lysine, and tyrosine are critical amino acids responsible for agglutinin activity. Present efforts are directed to t he design and synthesis of glucan analogues which may serve as affinit y inactivating agents of the lectin. Such glucan derivatives may be of value in studies on the role of the lectin in cariogenesis.