Endocytosis, recycling, and lysosomal delivery of brush border hydrolases in cultured human intestinal epithelial cells (Caco-2).
Lysosomes of intestinal epithelial cells in vivo and in culture display strong immunoreactivity with monoclonal antibodies against various brush border enzymes as visualized by immunoelectron microscopy. Novel subcellular fractionation procedures were developed to study, by the pulse-chase technique and by internalization assays, the pathway along which two microvillar hydrolases, sucrase-isomaltase and dipeptidylpeptidase IV, are transported to lysosomes in the differentiated colon adenocarcinoma cell line Caco-2. 7-9% of metabolically labeled sucrase-isomaltase of dipeptidylpeptidase IV were present in lysosomes after 7-8 h of chase as intact complex-glycosylated molecules. Appearance of these enzymes in lysosomes was biphasic. Endocytosis studies with radioiodinated antienzyme monoclonal antibodies (monovalent antigen-binding fragments) and by means of cell surface iodination revealed only slow transport of the enzymes to lysosomes at a low level. However, both enzymes were internalized with different efficiencies and recycled to the cell surface via endosomes. These results suggest that in Caco-2 cells a significant amount of newly synthesized sucrase-isomaltase and dipeptidylpeptidase IV is directly imported into lysosomes bypassing the brush border membrane.
Matter
K
K
Department of Pharmacology, University of Basel, Switzerland.
Stieger
B
B
Klumperman
J
J
Ginsel
L
L
Hauri
H P
HP
D000230:Adenocarcinoma; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003110:Colonic Neoplasms; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D004705:Endocytosis; D004848:Epithelium / Q000201:enzymology; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008871:Microvilli / Q000201:enzymology*
D006867:Hydrolases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4