Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells.

Abstract

A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [3SS]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.

Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells

“…In culture, this cell line spontaneously develops characteristics of mature enterocytes with functional brush border microvilli and apical hydrolases (Pinto et al, 1983;Hauri et al, 1985). Caco-2 was used extensively in studies on the organization and function of human intestinal cells (reviewed by Zweibaum et al, 1991).…”

“…With different protein and lipid compositions, these domains are strikingly different in ultrastructure. For example, the apical surface (brush border) contains peptidases and disaccharidases (Hauri et al, 1985;Pinto et al, 1983), the basolateral domain contains several peptide receptors involved in the control of intestinal hydroelectrolytic secretion (Laburthe & Amiranoff, 1990), and the tight junctions contain specific proteins. These cells are thought to be a convenient model for enterocytes and make an excellent model to study the cell biological processes of adhesion and bacterial invasion.…”

Mosaic expression of membrane peptidases by confluent cultures of Caco‐2 cells

“…On reaching confluency Caco-2 cells undergo differentiation into enterocyte-like cells and express many hydrolases on the apical brush border [3--61. These hydrolases include some disaccharidases, for example sucrase-isomaltase and lactase-phlorizin hydrolase [4], and numerous peptidases [6]. Using indirect immunofluorescent staining, the membrane peptidase dipeptidylpeptidase IV (DPPIV) has previously been shown to be homogeneously distributed on the surface of Caco-2 cells 14.73.…”

The Pathobiochemistry of Gastrointestinal Symptoms in a Patient with Niemann-Pick Type C Disease

“…The monoclonal mouse antibodies against sucrase (HBB2/ 614/88) and isomaltase (HBB3/705/60) (Hauri et al 1985) were generously provided by Prof. Dr. H. P. Hauri (Biocenter, Basel, Switzerland) and Prof. Dr. E. Sterchi (University of Bern, Bern, Switzerland). Monoclonal antibodies mlac6 and mlac10 (Maiuri et al 1991) for immunoblotting of lactase-phlorizin hydrolase were generously provided by Prof. Dr. D. Swallow (University College London, United Kingdom).…”