Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells.
Human lactase-phlorizin hydrolase (LPH) is a digestive enzyme that is expressed in the small intestinal brush-border membrane. After terminal glycosylation in the Golgi apparatus, the 230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPHbeta and the 100-kDa profragment (LPHalpha). Since LPHbeta is not transport-competent when it is expressed separately from LPHalpha in COS-1 cells, it was suggested that LPHalpha functions as an intramolecular chaperone. What happens to LPHalpha after cleavage is still unclear. To analyze and localize LPHalpha in polarized epithelial cells, wild type and tagged LPH were stably expressed in Caco-2 cells. In tagged LPH, a vesicular stomatitis virus epitope tag was inserted into the LPHalpha region. Wild type and tagged proteins were processed at similar rates, and both cleaved LPHbeta forms were expressed at the apical cell surface. Pro-LPH was recognized by antibodies against LPH, a profragment epitope and the vesicular stomatitis virus tag. LPHalpha alone, however, could not be recovered by these antibodies. Our data suggest that LPHalpha is degraded immediately after cleavage.
Ouwendijk
J
J
Department of Cell Biology and Histology, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Peters
W J
WJ
van de Vorstenbosch
R A
RA
Ginsel
L A
LA
Naim
H Y
HY
Fransen
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D019556:COS Cells; D018938:Caco-2 Cells; D018076:DNA, Complementary; D006801:Humans; D006868:Hydrolysis; D010694:Lactase-Phlorizin Hydrolase / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D014162:Transfection
D018076:DNA, Complementary; D018832:Molecular Chaperones; D010694:Lactase-Phlorizin Hydrolase