Transcellular processing of disulfide- and thioether-linked peroxidase--polylysine conjugates in cultured MDCK epithelial cells.
Horseradish peroxidase (HRP) was conjugated to nondegradable polycationic poly(D-lysine) (PDL) through either a thioether (HRP-S-PDL) or a disulfide (HRP-SS-PDL) linkage. The binding and transcytosis of these conjugates was studied in Madin-Darby canine kidney (MDCK) cell monolayers grown on 3-microns microporous polycarbonate filters. Conjugation of HRP to PDL with both linkages markedly increased the binding of this protein onto the cell monolayers. However, an enhancement of the transcellular transport of HRP in both apical-to-basal and basal-to-apical directions was observed only in HRP-SS-PDL, but not in HRP-S-PDL. HRP-SS-PDL transport was inhibited by colchicine and by 4 degrees C incubation. The transport of 14C-sucrose was not affected by the presence of conjugates. These results indicate that the transport of the conjugate across the cell monolayers was due to a transcellular process rather than to any leakage of the cell junction caused by polycations. The disulfide linkage between HRP and PDL was cleaved rapidly at the basal and, to a lesser extent, at the apical surface of the cell. Neuraminidase treatment decreased the binding of the conjugates onto the cell surface, but did not decrease the transcellular transport, suggesting that not all surface-bound conjugates were available for transcytosis. These results demonstrate that disulfide linkages can be cleaved during transcytosis in MDCK cells. The cleavage, however, occurs mostly at the binding site on the cell surface, which may prevent the cellular uptake of the intact conjugate.
Wan
J S
JS
Division of Pharmaceutics, University of Southern California School of Pharmacy, Los Angeles 90033.
Persiani
S
S
Shen
W C
WC
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D009439:Neuraminidase; D011107:Polylysine; D013395:Sucrose / Q000378:metabolism; D013440:Sulfides / Q000378:metabolism*; D013696:Temperature; D014462:Ultrafiltration
D003432:Cross-Linking Reagents; D004220:Disulfides; D013440:Sulfides; D011107:Polylysine; D013395:Sucrose; D006735:Horseradish Peroxidase; D009439:Neuraminidase; D003078:Colchicine