Proteolytic processing in a non-lysosomal compartment is required for transcytosis of protein-polylysine conjugates in cultured Madin-Darby canine kidney cells.
The transcytosis of horseradish peroxidase, as well as its poly(L-lys) and poly(D-lys) thioether conjugates, was investigated in Strain I Madin-Darby canine kidney (MDCK) cell monolayers grown on 0.4 microns pore size polycarbonate membranes in Costar Transwells. The 3 types of HRP had almost identical rates of transport during the first 2 hr of incubation. However, a significant increase of basal-to-apical transport was detected beginning at 3 hr only in Transwells containing the poly(L-lys) conjugate. This increase was inhibited by colchicine (2 microM) and by the Bowman-Birk protease inhibitor (0.1 mg/ml), but not by NH4Cl (10 mM) or chloroquine (0.1 mM). The increase was abolished either by prior trypsinization of the conjugate or by incubation at 4 degrees C. Ultrafiltration studies indicated that the transcytosed poly(L-lys) conjugate was smaller in size than the original conjugate. These results indicate that the conjugate was processed during transcytosis in a non-lysosomal proteolytic compartment, where its poly(L-lys) moiety was selectively degraded, allowing active peroxidase to be released into the apical medium.
Division of Pharmaceutics, University of Southern California, School of Pharmacy, Los Angeles 90033.
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D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004285:Dogs; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney; D008247:Lysosomes / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism*; D014357:Trypsin
D006735:Horseradish Peroxidase; D010544:Peroxidases; D010447:Peptide Hydrolases; D014357:Trypsin