Binding of salivary glycoprotein-secretory immunoglobulin A complex to the surface protein antigen of Streptococcus mutans

Abstract

The interaction between a surface protein antigen (PAc) of Streptococcus mutans and human salivary agglutinin was analyzed with a surface plasmon resonance biosensor. The major component sugars of the salivary agglutinin were galactose, fucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Binding of salivary agglutinin to PAc was calcium dependent and heat labile and required a pH greater than 5. Binding was significantly inhibited by N-acetylneuraminic acid and alpha2,6-linked sialic acid-specific lectin derived from Sambucus sieboldiana in a dose-dependent manner. Pretreatment of the salivary agglutinin with sialidase reduced the binding activity of the agglutinin to the PAc molecule. The agglutinin was dissociated into high-molecular-mass glycoprotein and secretory immunoglobulin A (sIgA) components by electrophoretic fractionation in the presence of 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Neither of the components separated by electrophoretic fractionation, high-molecular-mass glycoprotein or sIgA, bound to the PAc molecule. Furthermore, the high-molecular-mass glycoprotein strongly inhibited the binding of the native salivary complex to PAc. These results suggest that the complex formed by the high-molecular-mass salivary glycoprotein and sIgA is essential for the binding reaction and that the sialic acid residues of the complex play an important role in the interaction between the agglutinin and PAc of S. mutans.

FIG. 1

Native PAGE (A) and Western blot (B) analyses of human salivary agglutinin. (A) Salivary samples were subjected to native PAGE (3 to 15% polyacrylamide), and the gel was stained with silver. The molecular mass markers used were bovine serum albumin (67 kDa), catalase (232 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa). (B) Salivary components on the gel were electrophoretically transferred to a nitrocellulose membrane, and the membrane was reacted with rabbit antibody against human IgA(α). Lanes: 1, whole saliva (2 μg); 2, salivary agglutinin (1 μg).

FIG. 2

SDS-PAGE (A), Western blotting (B), and lectin blotting (C) analyses of human salivary agglutinin. (A) Salivary samples were suspended in SDS-PAGE nonreducing (1% SDS) or reducing (1% SDS, 1% 2-mercaptoethanol) buffer and heated at 100°C for 3 min. Samples were then subjected to SDS-PAGE (3 to 15% polyacrylamide), and the gels were stained with silver. The molecular mass markers used were α-lactalbumin (14.4 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), β-galactosidase (116 kDa), and myosin (212 kDa). Lanes: 1, nonreduced whole saliva (2 μg); 2, nonreduced salivary agglutinin (1 μg); 3, nonreduced sIgA (0.5 μg); 4, reduced whole saliva (2 μg); 5, reduced salivary agglutinin (1 μg); 6, reduced sIgA (0.5 μg). (B) Salivary components on the gel were electrophoretically transferred to a nitrocellulose membrane, and the membrane was reacted with rabbit antibody against human IgA(α). Lanes: 1, nonreduced salivary agglutinin (1 μg); 2, reduced salivary agglutinin (1 μg). (C) Salivary components on the gel were electrophoretically transferred to nitrocellulose membranes, and the membranes were reacted with biotinylated SSA (lanes 1 and 2) or biotinylated MAM (lanes 3 and 4). Lanes: 1 and 3, reduced salivary agglutinin (50 μg); 2 and 4, reduced sIgA (5 μg).

FIG. 3

Heat stability of salivary agglutinin (A) and the effect of pH on the binding of the agglutinin to rPAc (B). (A) After salivary agglutinin (25 μg/ml) was treated at 25 to 100°C for 15 min, the samples were subjected to BIAcore analysis. (B) Reactions were carried out with salivary agglutinin (25 μg/ml) in 10 mM potassium phosphate buffer (pH 4 to 7) containing 0.15 M NaCl and 1 mM CaCl₂ (○) and 10 mM Tris-HCl buffer (pH 7 to 9) containing 0.15 M NaCl and 1 mM CaCl₂ (•). The binding of salivary agglutinin to rPAc is expressed as RU determined by BIAcore assay. Values are given as the means ± standard deviations of triplicate assays.

FIG. 4

Dose-dependent inhibition of the binding of salivary agglutinin to rPAc by the sialic acid-specific lectins MAM (○) and SSA (•). Salivary agglutinin (25 μg/ml) was allowed to react with rPAc immobilized on a sensor chip in the presence of various amounts of lectin (0 to 100 μg/ml). The binding is expressed as RU determined by BIAcore assay. Values are given as the means ± standard deviations of triplicate assays. Single asterisk, P < 0.01; double asterisk, P < 0.001 (compared with control [no addition of lectin]).

FIG. 5

Binding of salivary components to rPAc. The binding to rPAc of high-molecular-mass glycoprotein and sIgA components separated from salivary agglutinin by electrophoretic fractionation, sIgA purified by using jacalin-agarose, and salivary lysozyme was determined by BIAcore assay. Values are given as the means ± standard deviations of RU in triplicate assays. Symbols: ○, salivary agglutinin; •, high-molecular-mass glycoprotein separated by electrophoretic fractionation; □, sIgA components separated by electrophoretic fractionation; ▪, sIgA purified by using jacalin-agarose; ▵, salivary lysozyme.

FIG. 6

Effects of salivary components on the binding of salivary agglutinin to rPAc. Salivary agglutinin (25 μg/ml) was allowed to react with rPAc immobilized on a sensor chip in the absence (control) or presence of various amounts of high-molecular-mass salivary glycoprotein separated by electrophoretic fractionation (•) or sIgA components separated by electrophoretic fractionation (□) (0 to 400 μg/ml). The binding is expressed as RU determined by BIAcore assay. Values are given as the means ± standard deviations of triplicate assays. Single asterisk, P < 0.01; double asterisk, P < 0.001 (compared with the control).