Protamine interaction with the epithelial cell surface

Abstract

We have previously reported that exposing cultured Madin Darby canine kidney (MDCK) cells to the polycation protamine (PRO) results in increased short-circuit current and decreased barrier integrity as measured by mannitol permeability and transepithelial electrical resistance. To further investigate the interaction of PRO with the surface of epithelial cells, we labeled PRO with [14C] with use of reductive alkylation. [14C]PRO bound to the cells in a biphasic pattern. Approximately 10% of the [14C]PRO was bound to the cells in the first 5 min, followed by an additional 10% that was bound over the next 25 min. No additional [14C]PRO bound to the cells after the initial 30 min. Binding of [14C]PRO was inhibited by “cold” PRO, which suggested specificity. Binding was also inhibited by polyanions, serum, and albumin, agents previously found to protect MDCK cells from PRO-induced injury. The binding of PRO to MDCK cells was not inhibited by incubation of the MDCK cells with neuraminidase, to remove surface sialic acid residues, or with heparinase, to remove surface heparan sulfate, even though metabolic labeling experiments demonstrated that neuraminidase decreased cell sialic acid and heparinase decreased cell heparan sulfate. Neuraminidase and heparinase offered no protection from PRO injury and had no effect themselves on mannitol permeability. Incubation of the cells with trypsin, however, blunted both the binding of PRO to the cells and the increase in mannitol permeability after exposure of the cells to PRO.(ABSTRACT TRUNCATED AT 250 WORDS)