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 reagents. 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 the lectin. A low molecular weight alpha-(1-->6)-glucan provided partial protection against the reagents which inactivated the protein, whereas 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, although N-bromosuccinimide, a reagent capable of oxidizing tryptophan and less selective than HNB, was a very effective inhibitor of the glucan-dependent cellular aggregation. In the latter case, alpha-(1-->6)-glucan did not protect. Hydroxylamine partially restored the loss of lectin activity due to treatment of the cells with N-acetylimidazole (highly specific for tyrosine), glycine methyl ester plus water-soluble carbodiimide (specific for carboxylates), and diethylpyrocarbonate (specific for histidine). Because the soluble form of GBL rapidly loses activity when purified, it was necessary to perform the chemical modification 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 because lectin activity could be restored in some cases, it was possible to identify likely essential amino acid residues needed for glucan binding. The results, taken together, suggest that aspartic (and/or glutamic) acid, histidine, lysine, and tyrosine are critical amino acids responsible for agglutinin activity. Present efforts are directed to the design and synthesis of glucan analogues which may serve as affinity inactivating agents of the lectin. Such glucan derivatives may be of value in studies on the role of the lectin in cariogenesis.
Singh
J S
JS
Academic Health Center, University of Louisville, KY 40292.
Taylor
K G
KG
Doyle
R J
RJ
D000596:Amino Acids; D001426:Bacterial Proteins; D001665:Binding Sites; D005936:Glucans / Q000378:metabolism* / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D016986:Streptococcus sobrinus / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D001426:Bacterial Proteins; D005936:Glucans; D006146:Guanidines; D007211:Indoles; D037102:Lectins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C082429:glucan-binding lectin, Streptococcus sobrinus; C030374:indole; D019791:Guanidine