Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells.
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 [35S]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.
D000911:Antibodies, Monoclonal; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004186:Disaccharidases / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism*; D007583:Jejunum / Q000201:enzymology; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D004186:Disaccharidases; D010447:Peptide Hydrolases