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MG2 and Lactoferrin Form a Heterotypic Complex in Salivary Secretions

¹ Departments of Periodontology and Oral Biology,
² Biochemistry, and
³ Medicine, Boston University Medical Center, 80 East Concord Street, K-312, Boston, MA 02118;

View larger version (31K): [in a new window]   Figure 1. Panning procedure. Step 1. The target protein (20 µg of MG2) was plated for 1 hr. Step 2. The plate was blocked for 1 hr. Step 3. We added the pFlitrx library to the plate to allow for interaction of expressed dodecapeptides on E. coli flagellae with bound target protein for 1 hr. Step 4. E. coli (unbound) that did not interact with immobilized target protein were washed off. Step 5. Bound cells were eluted by mechanical agitation. Step 6. Eluted cells were grown overnight at 37°C. The panning procedure was repeated 11 times.

View larger version (29K): [in a new window]   Figure 2. Compositional analysis of dodecapeptides. (A) The solid bars represent the expected percentage of each amino acid in dodecapeptides in the initial random library. (B) The solid bars represent the observed percentage of each amino acid in dodecapeptides in the sub-library obtained after 12-day panning with MG2.

View larger version (20K): [in a new window]   Figure 3. Western and Far-Western blots showing interactions between MG2 and lactoferrin. SMSL (50 µL) from two subjects was subjected to SDS-PAGE on 7.5% polyacrylamide gels (A) or to electrophoresis on pre-cast native 7.5% Tris-HCl gels (B), and proteins were transferred to nitrocellulose membranes. (A) Western blots were probed with anti-MG2 antibodies (lanes 1, 2) and anti-lactoferrin antibodies (lanes 3, 4). Far-Western blots were incubated with lactoferrin and probed with anti-lactoferrin antibodies (lanes 5, 6). Far-Western blots oxidized with periodate prior to incubation with lactoferrin were probed with anti-lactoferrin antibodies (lanes 7, 8). Far-Western blots pre-incubated with the synthetic lactoferrin peptide were then incubated with lactoferrin and probed with anti-lactoferrin antibodies (lanes 9, 10). (B) Western blots from native gels were probed with anti-MG2 antibodies (lanes 1, 2) and anti-lactoferrin antibodies (lanes 3, 4). In panel A, the dashed line shows the interface between the stacking and separating gels. There is no stacking gel in the native gel system, and the top of the gel is indicated by an arrow. Abbreviations: Western blot, W.B.; Far-Western Blot, F.W.B.; Anti-lactoferrin antibody, Anti-LF.