Implication of gliadin toxicity on actin integrity and subsequent protein trafficking in vitro

Celiac disease (CD) is a chronic inflammation of the small intestine due to ingestion of gluten in genetically predisposed individuals (HLA-DQ2/DQ8). While there has been great progress in some areas of CD, e.g. diagnostic, genetic aspects, or immunology, the molecular basis of the mucosal damage, especially at the outset of etiopathology, is poorly understood. It has been described that treatment with gliadin peptides leads to morphological alterations of the actin cytoskeleton in both biopsy specimens and Caco-2 cells. This points to a possible role of actin during early steps in the pathogenesis of CD, since actin is a key player in a variety of cellular events e.g. cell shape, protein transport and sorting, and endocytosis. For these reasons, the present study was aimed to elucidate the gliadin-induced rearrangements of the actin cytoskeleton on the molecular level and to investigate this effect on subsequent protein trafficking. Employment of discriminative experimental procedures revealed new insights into immediate effects on two different cell types (COS-1 and Caco-2) due to exposure with Frazer’s Fraction (FF), the source of gliadin peptides. Initial investigations with COS-1 cells that were treated with FF revealed morphological alterations in the actin filaments, which appeared as fragmented and aggregated structures in confocal laser microscopy. This observation indicates deterioration in the integrity of the actin cytoskeleton. Interestingly, there was a decrease of immunoprecipitated radio-active labeled actin, which suggests conformational changes. Leading thus to an altered antibody-binding capacity of actin. In contrast to FF, cytochalasin D (cytoD) induced a very drastic disruption of actin, which led to its accumulation in dense foci and patches concomitant with alteration in the cell shape. Moreover, actin was only partially reduced in immunoisolates from cytoD-treated cells. Therefore, the effect of FF appears to be different from that of cytoD. On the other hand, a peptic-tryptic digest of ovalbumin (OVA), that represents another food antigen, has no effect on actin neither in COS-1 cells nor in Caco-2 cells indicating that the effect on actin is gliadin-specific. Stimulation of Caco-2 cells with FF revealed also changes in the integrity of the actin network. In intestinal epithelial cells, the brush border is formed by a huge number of microvilli that are maintained by numerous actin filaments connected to the overlying brush border membrane (BBM) and the adjacent actin network. Isolation of BBMs from FF- stimulated Caco-2 cells showed a strong reduction of actin in the BBM-fraction which suggests a decrease in the association of actin with the BBM. Interestingly, α-tubulin displayed in turn an increase in the protein level indicating a compensational effect. The alteration of the actin cytoskeleton is presumably induced by direct interaction of FF-deriving gliadin peptides with actin or, more likely, actin binding proteins. Here, confocal imaging of FF-treated COS-1 cells showed a clear co-localization of the “„toxic“” gliadin peptide 31-49 with the actin cytoskeleton and co-immunoprecipitation revealed an increase of actin in the immunoisolates with time. On the other hand, Caco-2 cells displayed an alternating binding pattern of co-immunoprecipitated actin after prolonged stimulation up to 72h although the amount of actin in total lysates remains the same. A varying binding pattern together with the appearance of actin fragments in confocal images after FF-stimulation suggests a favored binding of gliadin peptides to the filamentous form of actin. The alterations of the actin cytoskeleton elicited impaired protein trafficking of intestinal sucrase-isomaltase (SI), a glycoprotein that follows an actin-dependent vesicular transport to the cell surface. This came along with reduced enzymatic function of SI in the BBM-fraction of Caco-2 cells. The transport of aminopeptidase N (ApN) and dipeptidylpeptidase IV (DPPIV) was only partially affected by FF. This is presumably due to their property to follow both a direct and a transcytotic pathway to the BBM. The latter pathway involves predominantly microtubules. By contrast, the actin-independent protein transport, as monitored by transport of the representative protein intestinal lactase phlorizin hydrolase (LPH), remains unaffected. In addition to anterograde transport processes, actin-dependent retrograde events are also found to be hampered by FF stimulation. Vesicles that are released from the trans-Golgi network do not only contain proteins but also lipids as SI associates with membrane microdomains enriched in cholesterol and sphingomyelin. Normally, these lipids are often internalized and recycle back to the plasma membrane. The establishment of an assay using biotinylated wheat germ agglutinin (WGA) provides an opportunity to assess the endocytic capability of treated and non-treated Caco-2 cells. The data revealed a substantially block of the endocytic pathway in FF-treated cells that led to an accumulation of cholesterol, and sphingolipids in the BBM. Altogether, the data demonstrate that FF deteriorates the actin cytoskeleton in COS-1 cells and Caco-2 cells by direct interaction with actin or, more likely, actin binding proteins leading thus to altered integrity. Consequently, this results in reduced protein trafficking and hampered endocytic events with subsequent alterations in the protein and lipid composition of the BBM. The reduced levels of the disaccharidase SI in the BBM suggest therefore a potential pathomechanism of carbohydrate malabsorption in CD.