Calcium-binding properties of SSP-5, the Streptococcus gordonii M5 receptor for salivary agglutinin.
Streptococcus gordonii M5 expresses a lectin on its surface (SSP-5) which binds to human salivary agglutinin (SAG). This interaction requires sialic acid residues of SAG and divalent cations and may mediate the colonization of oral tissues by this organism. In this report, we show that the binding of SAG to SSP-5 requires calcium and that SSP-5 is a high-affinity calcium-binding protein. SAG-mediated aggregation of S. gordonii M5 was inhibited by 1 mM EDTA, and the restoration of aggregation occurred only upon the readdition of calcium. To ascertain the level at which calcium exerts its effects, the calcium-binding properties of SSP-5 were evaluated by using a 45Ca binding assay. In addition, a kinetic analysis of calcium binding was carried out by using fura2, a fluorescent calcium-binding dye. These analyses showed that SSP-5 is a high-affinity calcium-binding protein that binds 1 mol of calcium per mol of protein and has a dissociation constant of 0.45 +/- 0.2 microM. The calcium-binding capacity of SSP-5 was also calculated independently to be 1.0 +/- 0.2 mol of Ca per mol of SSP-5 by column chromatography on Sephadex G-25 equilibrated with 10 microM 45Ca. To localize the calcium binding site of SSP-5, a series of C-terminal deletion mutants were expressed in Escherichia coli and evaluated for calcium-binding activity. Deletion of the 250 C-terminal residues of SSP-5 had little effect on calcium binding. However, deletion of residues 1168 to 1250 resulted in the loss of calcium-binding activity, suggesting that this region is important for calcium binding by SSP-5.
Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104.
Y
Y
Duan
E
E
Fisher
D
D
Malamud
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Golub
D R
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Demuth
D018829:Adhesins, Bacterial; D000373:Agglutinins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D010306:Parotid Gland / Q000737:chemistry; D011975:Receptors, Mitogen / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012463:Saliva / Q000737:chemistry*; D017384:Sequence Deletion; D013291:Streptococcus / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D018829:Adhesins, Bacterial; D000373:Agglutinins; D002135:Calcium-Binding Proteins; D011975:Receptors, Mitogen; D011994:Recombinant Proteins; C068465:salivary agglutinin receptor, Streptocococcus; D002118:Calcium