Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption.

Absorption of dietary fat in the small intestine is accompanied by a rise of intestinal alkaline phosphatase (IAP) in the serum and of secretion of IAP-containing surfactant-like particles from the enterocytes. In the present work, fat absorption was studied in organ cultured mouse intestinal explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A lipid analysis revealed that fat absorption caused a marked increase in the microvillar membrane contents of free fatty acids. In conclusion, fat absorption rapidly induces a transient clathrin-dependent endocytosis via coated pits from the enterocyte brush border. The process selectively internalizes IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles.

Antibodies in the small intestine: mucosal synthesis and deposition of anti-glycosyl IgA, IgM, and IgG in the enterocyte brush border.

Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and beta2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, beta2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that "anti-glycosyl" antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface.

Anti-glycosyl antibodies in lipid rafts of the enterocyte brush border: a possible host defense against pathogens.

The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens.

Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells.

The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.

Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism.

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.

Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes.

The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid 'rafts'. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo G(M1), and they were sensitive to cholesterol depletion by methyl-beta-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border.

Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts".

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.