Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

Abstract

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-β-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral β-galactoside residues of the characteristic GlcNAcα1→4Galβ1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions

“…(A) Distribution of MUC5AC (dark blue), MUC6 (lectin GSA-II; light blue), and TFF1 (black) after SEC on a Sephacryl S-500 HR column. The distribution of MUC5AC and MUC6 on this column has been reported previously [43]. Furthermore, the relative intensity of the hybridization signals (autoradiography and semi-quantitative analysis) obtained after incubating the blot with 125 I-TFF1 homodimer (overlay assay) is shown in red.…”

“…To confirm this unexpected result, in vitro binding of 125 I-labeled dimeric TFF1 was repeated with a human gastric extract separated via SEC on a HiPrep 16/60 Sephacryl S-500 High-Resolution (S-500) column ( Figure 6). We have shown previously that this column is able to separate MUC5AC and MUC6 [43]. The distribution of the bound 125 I-labeled homodimeric TFF1 resembles the distribution of MUC6, but not that of MUC5AC, supporting TFF1 homodimer interaction with MUC6 ( Figure 6A).…”

“…As we could previously separate MUC5AC and MUC6 by SEC on an S-500 column [43], here, we used just the same fractions for hybridization studies with 125 I-TFF1 homodimer ( Figure 6). The hybridization pattern confirmed that TFF1 bound to the mucin MUC6, but not MUC5AC ( Figure 6A).…”

Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

“…Given the known lectin activities of TFF modules [29][30][31][32], one could hypothesize that FIM-A.1 binds as a lectin to the carbohydrate moiety of another mucin, such as FIM-C.1. Alternatively, another mucin, such as the TFF modules of FIM-C.1, might bind the carbohydrate moiety of FIM-A.1.…”

“…The aim is to check whether the unusual mucins containing TFF domains (i.e., the short FIM-A.1 and the much-longer FIM-C.1) form a tight complex. To accomplish this, we used separation on a Sephacryl S-500 HR (S-500) column because, here, even the human mucins MUC5AC and MUC6 appear as different entities [32]. We also purified the unusually short FIM-A.1 via a combination of SEC and anion-exchange chromatography, labeled FIM-A.1 radioactively with 125 I, and performed first in vitro binding studies.…”