Biochemical Properties of Gastrokine-1 Purified from Chicken Gizzard Smooth Muscle

Abstract

The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties.BackgroundGNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells.Methodology/Principal FindingsFrom fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods.Conclusion/SignificanceAltogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

Gastrokine-2 is transiently expressed in the endodermal and endothelial cells of the maturing mouse yolk sac

“…Secretion of gastrokine-2 by YS endodermal cells is yet to be formally demonstrated. Gastrokine-1 protein associates with F-actin (Hnia et al, 2008). In contrast, Gkn2 protein was most found in the basal side of the YS endoderm, whereas F-actin in the YS is concentrated at the inner leaf of the apical cell membrane facing the maternal tissue suggesting that the majority of YS gastrokine-2 is not associated with F-actin.…”

“…Gastrokine-1 protein has been shown to associate with filamentous (F)-actin (Hnia et al, 2008). We therefore assessed the localisation of gastrokine-2 protein in the YS compared to F-actin as indicated by phalloidin binding ( Fig.…”

Ectopic expression of gastrokine 1 in gastric cancer cells up-regulates tight and adherens junction proteins network

“…Because chicken GKN1 has been proved to interact with cytoskeleton by exerting a cytoprotection action [21], we investigated the potential association of human GKN1 with actin in both gastric cancer cells and in solution. Compared to cancer gastric tissue (Fig.…”

“…This finding suggested that the N-terminal GKN1 sequence is important for its interaction with cellular compartments involved in the secretory mechanism. Hnia et al [21] showed that chicken GKN1 was well distributed throughout the gastric epithelium, but also colocalized and directly interacted with actin in smooth muscle cells probably through the presence of two or possibly three predicted trans-membrane helices. Also mature human GKN1, as predicted at TMpre Swiss Institute of Bioinformatics [25], was predicted as type II TM protein (N-terminus inside and 2 strong transmembrane helices F3-A20 and L158-V174), which might explain its prevalent membrane localization (Fig.…”

“…However, when exogenous protein was administered, GKN1 was localized at the membrane level in BGC-823 cells [20]. These results, together with data from bioinformatics analyses, suggest that GKN1 is a secretory protein that plays important roles in maintaining the physiological function of the gastric mucosa [1,14,16,17], and because normal stomach mucosa is in constant need of regeneration, it might also play an autocrine or paracrine role [16] and be involved in cytoskeleton protection [21]. However, to the best of our knowledge, no data are available on GKN1 subcellular localization in cancer experimental systems.…”