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Different Forms of TFF3 in the Human Saliva: Heterodimerization With IgG Fc Binding Protein (FCGBP)

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Different Forms of TFF3 in the Human Saliva: Heterodimerization With IgG Fc Binding Protein (FCGBP)

Till Houben et al. Int J Mol Sci.

Abstract

The peptide TFF3 is a member of a family of secretory lectins, and is typically synthesized by mucous epithelia together with mucins. It is mainly released from intestinal goblet cells as a high-molecular mass heterodimer with IgG Fc binding protein (FCGBP). Herein, we investigated human saliva by fast protein liquid chromatography (FPLC) and proteomics and identified high- and low-molecular-mass forms of TFF3. Whereas the high-molecular-mass forms represent a heterodimer with FCGBP, the low-molecular-mass forms represent homodimeric TFF3 forms. Proteomic analysis also revealed a C-terminally truncated form of TFF3. We hypothesize that salivary TFF3-FCGBP might play a role in the innate immune defense of the oral cavity and that TFF3 might also bind to microbial glycans. The known interaction of TFF3 with the agglutinin DMBT-1, a typical constituent of human saliva, further supports this protective role.

Keywords: DMBT1; FCGBP; IgG Fc binding protein; TFF3; lectin; mucin; saliva; trefoil factor.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of human saliva from a single individual (S-23). (A) Elution profile after SEC on a Superdex 75 HL column as determined by absorbance at 280 nm. Fractions positive after PAS staining are shown in pink. Underneath: Distribution of the relative TFF3 content in the fractions as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the typical 7k-band intensities (TFF3 monomer). (B) 15% SDS-PAGE and subsequent Western blot analysis of the high-molecular-mass fractions B7-B9. Samples were analyzed under reducing (R) and non-reducing conditions (NR), respectively, for their TFF3 immunoreactivity. The molecular mass standard is indicated on the left. (C) 15% SDS-PAGE and subsequent Western blot analysis of low-molecular-mass fractions C12/D1 and D5/D6. Samples were analyzed under reducing (R) and non-reducing conditions (NR), respectively, for their TFF3 immunoreactivity. The molecular mass standard is indicated on the left.
Figure 2
Figure 2
Analysis of high- and low-molecular-massfractions of human saliva of three individuals after SEC on Superdex 75 HL column. (A) 1% AgGE and subsequent Western blot analysis of the high- (B7) and low-molecular-mass fractions (C12), respectively, of the individuals 1–3. Shown are the reactivities for TFF3 and FCGBP, respectively. The dye bromophenol blue (BPB) is marked on the left. (B) 15% SDS-PAGE and subsequent Western blot analysis of the high-molecular-mass fractions B7 (after SEC on a Superdex 75 HL column) of the same three individuals as in (A). Samples were analyzed under reducing (R) and non-reducing conditions (NR), respectively, for their TFF3 immunoreactivity. The molecular mass standard is indicated on the left. (C) 15% SDS-PAGE and subsequent Western blot analysis of the low-molecular-mass fractions C12 analogous to (B).
Figure 3
Figure 3
Purification of different forms of TFF3 from saliva and characterization by LC-ESI-MS/MS analysis. (A) Saliva of a single individual (S-20) was purified via SEC on a Superdex 75HL column (analogous to Figure 1) and then the low-molecular-mass region (about fractions C8-D5) subsequently separated by anion exchange chromatography on a Resource Q6 column. Shown is the distribution of the relative TFF3 immunoreactivity in the fractions as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the typical 7k-band intensities (monomeric TFF3). Fractions B8-C3 were concentrated, desalted and further analyzed. (B) 15% SDS-PAGE under non-reducing conditions of fractions B11–C2 (see (A)) and subsequent Western blot analysis concerning TFF3 (marked are bands 1-3). (C) Separation of combined fractions B8-C3 (see (A)) by non-reducing 15% SDS-PAGE followed by Coomassie staining. Marked are the bands excised (1, 2, 3) and subjected to Western blot analysis under reducing conditions or LC-ESI-MS/MS analysis. (D) Separation of the excised bands 1, 2, and 3 (see (C)) by 15% SDS-PAGE under reducing conditions and Western blot analysis concerning TFF3. For comparison, a human colon extract is shown (lane c). The star marks a non-specific band in lane 3 recognized by the anti-TFF3 antiserum. (E) Results of the LC-ESI-MS/MS analysis after tryptic in-gel digestion of the bands 1, 2, and 3, respectively. Identified tryptic peptides belonging to TFF3 are highlighted in red.

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