Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells.
The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.
Zhang
Y
Y
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Wick
D A
DA
Haas
A L
AL
Seetharam
B
B
Dahms
N M
NM
D001483:Base Sequence; D002412:Cations; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D015870:Gene Expression; D005966:Glucuronidase / Q000032:analysis; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000648:ultrastructure; D037181:Lectins, C-Type; D008247:Lysosomes / Q000201:enzymology*; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008361:Mannosidases / Q000032:analysis; D008969:Molecular Sequence Data; D012313:RNA / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics; D014452:Ubiquitins / Q000378:metabolism*; D043323:alpha-Mannosidase; D001616:beta-Galactosidase / Q000032:analysis; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis
D002412:Cations; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface; D014452:Ubiquitins; D012313:RNA; D007335:Insulin-Like Growth Factor II; D008361:Mannosidases; D001616:beta-Galactosidase; D043323:alpha-Mannosidase; D005966:Glucuronidase; D001619:beta-N-Acetylhexosaminidases