The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions

Abstract

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier

“…This hypothesis is in line with observations indicating at least partial non-covalent binding of Tff3 and Fcgbp in the murine duodenum [12] and human colon [23]. Furthermore, a non-covalent interaction would be comparable with the non-covalent binding of the lectin TFF2 to porcine or murine gastric mucin MUC6 [38,39]. Maybe fucose residues in the carbohydrate moiety of FCGBP (Figure 6) play a role for a possible lectin binding of TFF3.…”

“…Extraction and fractionation by SEC of endocervical specimens were similarly carried out as previously described [39]. Generally, 0.6 to 1.0 g tissue was minced with a scalpel and extracted with a 10-to 15-fold amount (w/v) of buffer (30 mM NaCl, 20 mM Tris-HCl pH 7.0 plus protease inhibitors) in a Precellys ® 24 lyser/homogenizer as described (aqueous extracts: supernatant E1) [39]. Then, 5 mL of the extracts E1 were fractionated by SEC with the ÄKTA TM FPLC system (Amersham Biosciences, Freiburg, Germany) as described (fraction numbering: A1-A12, B1-B12, etc.)…”

“…Then, 5 mL of the extracts E1 were fractionated by SEC with the ÄKTA TM FPLC system (Amersham Biosciences, Freiburg, Germany) as described (fraction numbering: A1-A12, B1-B12, etc.) using a HiLoad 16/600 Superdex 75 prep grade column (S75HL; 20 mM Tris-HCl pH 7.0, 30 mM NaCl plus protease inhibitors; flow rate: 1.0 mL/min; 2.0 mL fractions) [39]. The fractions were stored at −20 • C.…”

“…Denaturing vertical SDS-PAGE under reducing and non-reducing conditions, respectively, protein staining with Bio-Safe Coomassie Stain G-250 without fixation, nondenaturing horizontal AgGE, periodic acid-Schiff (PAS) staining of mucins (dot blot), and Western blot analysis (classical electrophoretic transfer after SDS-PAGE and capillary blotting after AgGE, respectively), including staining with Amidoblack, were described previously [23,25,39,72]. When indicated, gels after non-reducing SDS-PAGE were subjected to post-in-gel reduction with 1% mercaptoethanol at 50 • C for 2 min according to a previous report [39]. In addition, 1% AgGE was used to separate high-molecular-mass proteins under non-denaturing conditions [73].…”

“…FCGBP was shown with PAP389Hu01 (Cloud-Clone Corp., Katy, TX, USA) against amino acids 5176-5344 of human FCGBP. Detection of the mucin MUC6 was carried out with the biotinylated lectin GSA-II from Griffonia simplicifolia as reported [39,74]. Fucosylation was analyzed using the biotinylated lectin AAA from A. anguilla (Vector Laboratories, Biozol Diagnostica Vertrieb GmbH, Eching, Germany).…”