TNF-alpha release by monocytic THP-1 cells through cross-linking of the extended V-region of the oral streptococcal protein I/II.

We tested the hypothesis of a conserved activation mode of monocytic THP-1 cells by proteins I/II expressed by several species of oral streptococci through the specific role of the extended V-region. We studied the binding and modulating activities of six proteins I/II purified from strains representing four different species of oral streptococci, and of expression products of polymerase chain reaction-amplified sequences encoding corresponding extended V-regions. We found that the different proteins I/II bound to THP-1 cells in a sugar-dependent mode involving the extended V-region. Furthermore, all the proteins I/II stimulated THP-1 cells to produce tumor necrosis factor alpha, indicating that these properties are not strain- or species-specific. Despite the weak stimulation of THP-1 cells by the extended V-region alone, we obtained evidence that cross-linking of this region can be one of the mechanisms involved in monocytic cell activation by proteins I/II.

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor alpha in the monocyte cell line THP-1.

The induction of tumour necrosis factor (TNF)-alpha from the monocytic cell line THP-1 by the streptococcal antigen I/II from Streptococcus mutans serotype f (protein I/IIf) was studied by use of recombinant polypeptides containing the discrete domains of the protein. The derivatives carrying the N-terminal alanine-rich region (A region) and the adjacent variable region (extended V region) of the protein bound to THP-1 cell extracts in a saturable fashion, and one derivative lacking both the A and the extended V regions was not able to bind monocyte cell extracts, suggesting that the domains responsible for the binding of protein I/IIf to monocytes were the A and the extended V regions. Sodium metaperiodate pretreatment of THP-1 cell extracts, tunicamycin pretreatment of monocyte cells or competition with N-acetyl neuraminic acid (NANA) and fucose resulted in a 45-70% reduction in binding activity of the derivatives carrying the extended V region, demonstrating the lectin-like mode of recognition of the monocytic receptor by the extended V region and the role of NANA and fucose in this recognition process. Besides, the stimulation of monocytes to release TNF-alpha by the derivatives containing the A region and the extended V region was effective and was not affected by the addition of polymyxin B or vitamin D3, suggesting that CD14 does not play the role of receptor in stimulation of monocytes by protein I/IIf to release TNF-alpha.

The N-terminal half part of the oral streptococcal antigen I/IIf contains two distinct binding domains.

In order to investigate the binding properties of the antigen I/IIf from Streptococcus mutans, we analyzed the binding activity of five I/IIf derivatives expressed by I/IIf gene derivatives obtained by insertion of a kanamycin resistance marker. ELISA-derived binding assays showed that the derivatives containing both the N-terminal alanine-rich domain (A-region) and an A-region distal domain extending to amino-acid 766 were the most effective in binding biotinylated (Biot-) human salivary components (SAC) and Biot-epithelial cell membrane components. Sodium metaperiodate treatment of SAC inhibited these interactions, suggesting a binding specificity of the A-region distal domain for carbohydrate residues. All the I/IIf derivatives were found to bind Biot-type I collagen, Biot-laminin, Biot-keratin, and Biot-fibronectin, the derivatives containing the A-region but lacking the A-region distal domain exhibiting the highest binding levels. Sodium metaperiodate treatment of laminin had no effect on its binding to the derivatives, suggesting that carbohydrate residues of the ligand were not involved.