Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action.

Abstract

Using anticholeragen antibodies and '25 1-protein A, we developed a specific and quantitative assay for measuring choleragen on the surfaces of cultured cells. When neuroblastoma cells containing bound toxin were incubated at 37°C, surface toxin disappeared with a half-life of -2 h and a significant loss was detected by 10 min. When cells were incubated with ' 25 1-choleragen in order to measure toxin degradation, cell-associated radioactivity disappeared with time and a corresponding amount of TCA-soluble label appeared in the culture medium with a half-life of 4-6 h. No degradation was detected until 45 min. Although there was a lag of 15 min before bound choleragen activated adenylate cyclase, the enzyme became maximally activated between 45 and 60 min . Similar results were obtained with Friend erythroleukemia cells. Internalization, degradation, and activation all were blocked when the cells were maintained at 4°C. At 22°C, internalization and activation occurred, albeit at a slower rate, whereas degradation was effectively inhibited . These results indicated that choleragen does not have to be degraded by intact cells in order for it to activate adenylate cyclase. Some internalization of the toxin, however, appears to precede the activation process.Choleragen (CT), the active agent of cholera, is composed of two components, A and B (4) . The B component binds to specific receptors on the cell surface that are believed to be the ganglioside GM, (see reference 6 for a recent review) . The A component consists of two polypeptide chains, A l and A2, connected by a single disulfide bond. The A l peptide is an ADP-ribosyltransferase (23) and catalyzes the transfer of ADPribose from nicotinamide adenine dinucleotide (NAD) to the regulatory component of adenylate cyclase (2, 15). This modification results in a persistent activation of adenylate cyclase . With intact cells, there is a definite lag period before cyclase activity begins to rise in response to the toxin (1, 5). When membranes are incubated with A 1 plus NAD, there is no lag (14). It is believed that during the lag period CT or some part of it is translocated across the membrane, that Al is formed, and that adenylate cyclase is then activated by A l (6, 9, 12, 16). Several groups, using immunochemistry or cytochemistry and electron microscopy, have reported that CT becomes internalized in a time-and temperature-dependent manner (17,19,20). These observations, however, were not correlated with the activation of adenylate cyclase . Other groups have suggested that CT must first be degraded by the cell in order to generate a fragment that can then activate the cyclase (18,21,22) .The present studies were initiated in order to develop a specific and quantitative method for assaying surface bound CT and to follow the kinetics of CT internalization, degradation, and activation of adenylate cyclase . MATERIALS AND METHODS Cells and Cell CultureMouse neuroblastoma clone NB41A3 was obtained from the American Type Culture Collection (Rockville, MD) and ...

Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface

“…However, in a migratory situation, speed may be an issue. It has been shown previously that internalisation by caveolae is two to four times slower that clathrin-mediated endocytosis (Fishman, 1982;Tran et al, 1987). Clathrin-mediated and caveolae-dependent pathways may be involved in the differential localisation of internalisation at the cell surface.…”

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann–Pick C1-deficient cells

“…First, the toxin is recycled back to the PM where it is excreted in its intact form. Of note, the intact form of CT in the medium can be separated from degradation products by TCA precipitation (19). Second, the toxin can enter a retrograde transport pathway that leads to the Golgi apparatus and eventually to the ER.…”

“…To examine whether the retrograde transport pathway that leads to the Golgi apparatus͞ER is also altered, we analyzed cell-associated fractions by gel filtration chromatography (19). As expected, there were no free A subunits in both wt and NPC1(Ϫ) cells at the end of the 4°C incubation.…”

“…Gels were processed for autoradiography and developed with BAS2000 (Fujitsu, Tokyo). Radioactive proteins in the cell-associated fractions were analyzed by gel filtration chromatography as described (19). Briefly, cells were recovered in ice-cold PBS͞10 mM N-ethylmaleimide.…”

“…Intracellular transport of 125 I-CT was assayed as described (19,20), with some modifications. Cells were incubated at 4°C for 1 h in the reaction medium (F12͞25 mM Hepes, pH 7.4͞0.01% BSA) containing 10 nM 125 I-CT, and after washing they were further incubated in reaction medium at 37°C.…”