Salivary Agglutinin, Which Binds Streptococcus mutansand Helicobacter pylori, Is the Lung Scavenger Receptor Cysteine-rich Protein gp-340

Akraporn Prakobphol,Feng Xu,Van M Hoang,Thomas Larsson,Jorgen Bergstrom,Ingegerd Johansson,Lars Frängsmyr,Uffe Holmskov,Hakon Leffler,Christina Nilsson,Thomas Borén,Jo Rae Wright,Nicklas Strömberg,Susan J Fisher

doi:10.1074/jbc.m006928200

Abstract

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Highlights

Able barrier that coats oral surfaces [5,6,7,8], and regulation of the composition of the oral flora
Isolation of Agglutinin for Mass Spectrometry Analyses—The salivary agglutinin was isolated by two methods
Whole parotid saliva was electrophoretically separated on a SDS-polyacrylamide gel, and the band corresponding to the molecular mass of the salivary agglutinin was excised (Fig. 1A, arrowhead)

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals, unless otherwise noted, were obtained from Sigma. Nitrocellulose membranes (0.45 ␮m) were obtained from Schleicher & Schuell. Mass Spectrometry of Peptides Isolated from the Biochemically Purified Agglutinin—The excised agglutinin band was macerated in a solution of high purity trypsin (0.05 ␮g/␮l in 25 mM ammonium bicarbonate). Aggregation of Bacterial Cells by Purified Agglutinin—The ability of affinity-purified parotid agglutinin to agglutinate a panel of streptococci (Table II) and H. pylori strain CCUG17875 was assessed as described earlier [20]. Binding of Agglutinin to Bacterial Cells—4 ml of either an H. pylori strain CCUG17875 or an S. mutans Ingbritt cell suspension (2 ϫ 109 cells/ml PBS) was incubated with an equal volume of fresh parotid saliva diluted 1:1 in PBS (experimental) or PBS alone (control). The cells were pelleted by centrifugation, washed twice with PBS, and suspended in 150 ␮l of SDS-PAGE loading buffer. An aliquot of the supernatant (ϳ25 ␮l) was subjected to SDS-PAGE and immunoblotting with anti-gp-340-1 as described above

RESULTS

Peptide sequence

DISCUSSION

Subgingival plaque