Properties of volkensin, a toxic lectin from Adenia volkensii

Volkensin, a highly toxic protein from the roots of Adenia volkensii (kilyambiti, kinoria), was purified by affinity chromatography on acid-treated Sepharose 6B. The toxin is a glycoprotein (Mr 62,000, neutral sugar content 5.74%) consisting of an A subunit (Mr 29,000) and of a B subunit (Mr 36,000) linked by disulfide and noncovalent bond(s). The amino acid, amino sugar, and neutral sugar composition of the protein were determined. Volkensin is a galactose-specific lectin and is a potent inhibitor of eukaryotic protein synthesis in whole cells as well as in a cell-free system (a rabbit reticulocyte lysate). The inhibitory and the lectin activities are functions of the A and B subunits, respectively. Volkensin can be included amongst the ricin-like toxins and resembles most closely modeccin, the toxin of Adenia digitata.     

Regulation of Na+/H+ and Cl-/HCO3- antiports in Vero cells

The effect of serum, phorbol-12-myristate-13-acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na(+)-coupled and Na(+)-independent Cl-/HCO3- antiport was studied in Vero cells by measuring 22Na+ and 36Cl- fluxes and changes in cytosolic pH (pHi). The Na(+)-independent Cl-/HCO3- antiport, which acts as an acidifying mechanism, is strongly pH-sensitive. In serum-starved cells it is activated at alkaline cytosolic pH, with a half-maximal activity at pHi approximately 7.20. Incubation with serum increased the activity of the Na(+)-independent Cl-/HCO3- antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+/H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (greater than 45 min) decreased its activity. The activity of the Na(+)-coupled Cl-/HCO3- antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short-term incubation with serum (less than 10 min) but decreased after prolonged incubation (greater than 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi-regulating antiports was a slight decrease in intracellular pH.     

Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Δg _m , was initially determined. DT-AB induced a ∼10-fold lower Δg _m than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg _m , whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg _m . Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Δg _m and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel. Received: 20 June 1996/Revised: 8 November 1996     

GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.     

Acidification of the cytosol inhibits endocytosis from coated pits

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.     

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

The rates of bicarbonate-dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl-. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate-linked uptake of 22Na+ was dependent on internal Cl- but not on internal Na+. At a constant external concentration of HCO3-, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO3- decreased and vice versa, which is compatible with the possibility that the ion pair NaCO3- is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl- both in the absence and presence of Na+. At alkaline internal pH, HCO3- stimulated the efflux of 36Cl- from preloaded cells, while at acidic internal pH both Na+ and HCO3- were required to induce 36Cl- efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.