Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography

Abstract

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.

Saliva-Bacterium Interactions in Oral Microbial Ecology

“…Immune sera and Fab fragments against Antigen B, but not Antigen A, inhibited the binding of S. mutans cells to sHA (Douglas and Russell, 1984b). Antigen B, which appears to be related to components subsequently named antigen I/II (Russell et al, 1980), P1 (Forester et al, 1983), PAc (Koga et al, 1990), SR (Ackermans et al, 1985b(Ackermans et al, , 1988Ogier et al, 1990), and SpaA (Abiko et al, 1989), was found to bind to a greater extent to parotid salivacoated enamel than to uncoated enamel (Russell and Rahemtulla, 1989). A correlation was found between the expression of this protein on the surface of a number of strains of mutans streptococci and adhesion to HA coated with the parotid agglutinin (Brady et al, 1992).…”

Cloning of the saliva-interacting protein gene from Streptococcus mutans

“…The molecular weight of the fusion peptide was consistent with the expression of all or almost all of the cloned sequence. However, a molecular weight of about 60,000 was far from 74,000, which is the molecular weight of the S. mutans 74K SR protein (2), and a true determinants on the fusion peptide synthesized in cells containing pSAD2-4 in ELISA experiments (Fig. 4).…”

“…Rabbit polyclonal antibodies and rat monoclonal antibodies raised against the S. mutans OMZ175 74K SR protein were obtained as described by Ackermans et al (1,2). Rabbit anti-S. mutans lactate dehydrogenase and anti-CWE antibodies were prepared as previously described (33,34).…”

“…CWE used as an antigenic reference was obtained from washed S. mutans OMZ175 cells (33), The 74K SR protein was purified from this extract as described by Ackermans et al (2).…”

“…Polyclonal antibodies against SpaA can partially block the adherence of S. mutans to saliva-coated hydroxyapatite beads (8). Purified 74K SR protein has the ability to inhibit the interactions between S. mutans cells and salivary glycoproteins (2). In a previous study, we described monoclonal antibodies against the S. mutans serotype f 74K SR protein.…”